Effect of heat on synthesis of gelatinases and pro-inflammatory cytokines in equine tendinocytes

Abstract

The aim of this study was to clarify whether matrix metalloproteinases (MMP-2 and -9: gelatinases) and pro-inflammatory cytokines [tumor necrosis factor (TNF) alpha and interleukin (IL)-1beta] are induced by heat in tendon tissue in vitro and to test the hypothesis that heat exposure causes tendinocytes to synthesize pro-inflammatory cytokines and that synthesis of these cytokines, in turn, leads to up-regulation of synthesis of gelatinases. Isolated tendinocytes from equine superficial digital flexor tendons were cultured and all experiments were performed on cells passaged 3 or 4 times. In the cells exposed to heat (37 to 45 degrees C, 0 to 60 min), the survival rate decreased sharply in a temperature- and time-dependent manner, especially at 42 and 45 degrees C. Cells exposed at 40 degrees C, however, showed little change in survival rate and morphology. Gelatin zymograms revealed that proMMP-2 and -9 were the only two MMPs remaining in the supernatant of the cultured tendinocytes, including that of untreated cells. Addition of TNFalpha and IL-1beta to the culture medium of tendinocytes accelerated proMMP-9 synthesis considerably. Heating the tendinocytes (40 degrees C) led to a three-fold increase in proMMP-9 synthesis in a short time. Only TNFalpha was detected in tendinocytes after heat exposure for 30 and 60 min. In contrast, IL-1beta was under the detectable level in ELISA. Cooling of heat-exposed cells from 40 degrees C to 37 degrees C considerably down-regulated cellular proMMP-9 synthesis. Furthermore, proMMP-9 level was greatly reduced in cells treated at lower temperatures, 20 degrees C and 5 degrees C. These findings support our hypothesis that hyperthermia in the horse tendon induces tendinocytes to synthesize pro-inflammatory cytokines and that the synthesis of these cytokines results in the up-regulation of gelatinases.