IL–1b SENSITIZES HUMAN ANNULUS CELLS TO SHEAR STRESS

Michelle Elfervig,
Joe Minchew, MD,
Eric Francke,
Mari Tsuzaki,
Albert Banes
UNC School of Medicine Chapel Hill, NC, USA

INTRODUCTION:
The etiology(ies) of spinal deformity and the factor(s) contributing to progression are not understood, particularly for adult degenerative scoliosis. Disc degeneration and altered mechanical loading are implicated as contributors to deformity development. Biomechanical and biochemical factors play a role in degeneration. However, the interaction between biochemical and biomechanical stimulation of disc cells has received limited study. Shear is a component of loading in the spine, especially in the asymmetric loading of deformity. IL–1b, detected in degenerative discs, is known to have effects on disc cell metabolism. We hypothesized an interaction between shear stress and IL–1b in human annulus cells. To evaluate this interaction, we measured the effect of combined stimuli on intracellular calcium concentration, [Ca2+]ic and signaling.

METHODS:
Annular tissue was obtained at surgery for scoliosis release, disc herniation or trauma. Cells were isolated and cultured to a non–dividing state. To test [Ca2+]ic, cells were plated on collagen–bonded Culture Slipsä for use with a FlexFloä shear stress device (Flexcell Intl. Corp) then incubated with 5 mM FURA–2AM, a calcium sensitive dye. Emission data were collected with a fluorescence microscope. Cells were incubated for 30 min in 1 nM IL–1b. Cell response to fluid– induced shear stress was tested at 1, 3, 5, 10, 15, 20 and 25 dynes/cm2 using the FlexFLoä. Differences in [Ca2+]ic, were compared using ANOVA and Tukey test.

RESULTS:
Human annulus (hAN) cells responded to isolated fluid–induced shear with a rise in [Ca2+]ic at 15 dynes/cM2 or above. IL–1b alone also produced a small, transient stimulation. hAN cells pretreated with IL–1b responded to shear at 5 dyne/cm2 and with a more dramatic and sustained increase in [Ca2+]ic when compared to shear then IL–1b or shear and IL–1b alone (p<0.001 for all comparisons). After [Ca2+]ic returned to baseline, cells could be restimulated by shear.

CONCLUSIONS:
To our knowledge, this is the first study documenting synergism of a signaling response to biomechanical and biochemical stimuli in disc cells. The effect of IL–1b on the [Ca2+]ic response of sheared cells was clearly more than additive. IL–lb treatment appeared to "sensitize" annulus cells to mechanical load. This increased susceptibility to mechanical load in the face of inflammatory cytokines may have implications as to the initiation and progression of degenerative deformity, particularly scoliosis.