Glial cell line-derived neurotrophic factor influences proliferation of osteoblastic cells.

Little is known about the role of neurotrophic growth factors in bone metabolism. This study investigated the short-term effects of glial cell line-derived neurotrophic factor (GDNF) on calvarial-derived MC3T3-E1 osteoblasts. MC3T3-E1 expressed GDNF as well as its canonical receptors, GFRα1 and RET. Addition of recombinant GDNF to cultures in serum-containing medium modestly inhibited cell growth at high concentrations; however, under serum-free culture conditions GDNF dose-dependently increased cell proliferation. GDNF effects on cell growth were inversely correlated with its effect on alkaline phosphatase (AlP) activity showing a significant dose-dependent inhibition of relative AlP activity with increasing concentrations of GDNF in serum-free culture medium. Live/dead and lactate dehydrogenase assays demonstrated that GDNF did not significantly affect cell death or survival under serum-containing and serum-free conditions. The effect of GDNF on cell growth was abolished in the presence of inhibitors to GFRα1 and RET indicating that GDNF stimulated calvarial osteoblasts via its canonical receptors. Finally, this study found that GDNF synergistically increased tumor necrosis factor-α (TNF-α)-stimulated MC3T3-E1 cell growth suggesting that GDNF interacted with TNF-α-induced signaling in osteoblastic cells. In conclusion, this study provides evidence for a direct, receptor-mediated effect of GDNF on osteoblasts highlighting a novel role for GDNF in bone physiology.

Differing mechanisms of leukocyte recruitment and sensitivity to conditioning by shear stress for endothelial cells treated with tumour necrosis factor-alpha or interleukin-1beta.

The cytokines tumour necrosis factor-alpha (TNFalpha) and interleukin-1beta (IL-1B) induce endothelial cells to recruit leukocytes. However, the exact adhesion and activation mechanisms induced by each cytokine, and their relative sensitivities to modulation by endothelial exposure to shear stress remain unclear. We cultured human umbilical vein endothelial cells (HUVEC) in glass capillaries at various shear stresses, with TNFalpha or IL-1B added for the last 4 h. Subsequently, human neutrophils were perfused over the HUVEC, and adhesion and migration were recorded. Both cytokines induced dose-dependent capture of neutrophils. However, while conditioning of HUVEC by increasing shear stress for 24 h diminished their response to TNFalpha, the response of HUVEC to IL-1B was similar at all shear stresses. The differing sensitivities were evident at levels of adhesive function and mRNA for adhesion molecules and chemokines. Analysis of nuclear factor kappaB (NF-kappaB)/Rel family of transcription factors showed that their expression and activation were modified by exposure to shear stress, but did not obviously explain differential responses to TNFalpha and IL-1B. Antibodies against selectins were effective against capture of neutrophils on TNFalpha-treated but not IL-1B-treated HUVEC. Stable adhesion was supported by beta2-integrins in each case. Activation of neutrophils occurred dominantly through CXC-chemokine receptor 2 (CXCR2) for TNFalpha-treated HUVEC, while blockade of CXCR1, CXCR2 and of platelet-activating factor receptors caused additive inhibition of migration on IL-1B-treated HUVEC. The mechanisms which underlie neutrophil recruitment, and their modulation by the haemodynamic environment, differ between cytokines. Interventions aimed against leukocyte recruitment may not operate equally in different inflammatory milieu.

Methods for exposing multiple cultures of endothelial cells to different fluid shear stresses and to cytokines, for subsequent analysis of inflammatory function.

Endothelial cells are conditioned by physicochemical environmental factors, including shear stress applied by flowing blood. However, the effects of shear conditioning on the functional responses of endothelial cells, such as ability to recruit leukocytes, remain uncertain. Here we describe a system for culturing multiple samples of endothelial cells under flow for prolonged periods, either at different shear stresses, or exposed concurrently to different concentrations of cytokines, for instance, tumour necrosis factor-alpha (TNF). The endothelial cells were cultured in glass capillaries (microslides) that could be conveniently transferred to a flow-based adhesion assay, to test the ability of the cultures to support adhesion and migration of flowing leukocytes. Paired control, 'static' samples were exposed to the identical medium and culture geometry. We found that the type of tubing used in the culture flow circuit and its maintenance at 37 degrees C were critical design factors, which could influence the response to TNF of the static controls which were exposed to recirculated medium. Endothelial cells conditioned by culture under flow showed a reduction in response to TNF, as judged by ability to induce the capture and migration of neutrophils. We found that the higher the shear stress, the weaker the ability to recruit neutrophils. This sensitivity to shear stress was greater if the cells were allowed to stabilise under static conditions for 24 h, compared to cells exposed to flow immediately after seeding. The inhibition of neutrophil recruitment was similar for cultures exposed to steady flow or flow with a pulsatile element (flow oscillation approximately 20% about the mean). Thus, we have developed a versatile culture system which allows investigations of functional modifications of endothelial cells and demonstrates the potential sensitivity of inflammatory responses to the local fluid environment.

Exposure to fluid shear stress modulates the ability of endothelial cells to recruit neutrophils in response to tumor necrosis factor-alpha: a basis for local variations in vascular sensitivity to inflammation.

Vascular endothelial cells are able to sense changes in the forces acting on them and respond, for instance, by modifying expression of a range of genes. However, there is little information on how such responses are integrated to modify homeostatic functions. We hypothesized that different shear stresses experienced in different regions of the circulation might influence endothelial sensitivity to inflammatory stimuli. We cultured human endothelial cells in tubes and exposed them for varying periods to shear stresses ranging from those typically found in postcapillary venules to those in arteries. When tumor necrosis factor-alpha was included in the flow cultures, we found startling differential effects of shear stress on the ability of endothelial cells to induce adhesion and migration of flowing neutrophils. Compared with static cultures, endothelial cells cultured at low shear stress (0.3 Pa) captured similar numbers of neutrophils but failed to induce their transendothelial migration. After exposure of endothelial cells to high shear stress (1.0 or 2.0 Pa), capture of neutrophils was largely ablated. The modification in response was detectable after 4 hours of exposure to flow but was much greater after 24 hours. From analysis of gene expression, loss of capture or migration was attributable to reduction in tumor necrosis factor-induced expression of selectins or CXC-chemokines, respectively. Thus, conditioning of endothelial cells by different flow environments may underlie variations in susceptibility to inflammation between different tissues or parts of the vascular tree.