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-infl ammatory cytokines [tumor necrosis factor (TNF) α and interleukin (IL)-1β] are induced by heat in tendon tissue in vitro and to test the hypothesis that heat exposure causes tendinocytes to synthesize pro-infl ammatory cytokines and that synthesis of these cytokines, in turn, leads to up-regulation of synthesis of gelatinases. Isolated tendinocytes from equine superfi cial digital fl exor tendons were cultured and all experiments were performed on cells passaged 3 or 4 times. In the cells exposed to heat (37 to 45°C, 0 to 60 min), the survival rate decreased sharply in a temperature-and time-dependent manner, especially at 42 and 45°C. Cells exposed at 40°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 TNFα and IL-1β to the culture medium of tendinocytes accelerated proMMP-9 synthesis considerably. Heating the tendinocytes (40°C) led to a three-fold increase in proMMP-9 synthesis in a short time. Only TNFα was detected in tendinocytes after heat exposure for 30 and 60 min. In contrast, IL-1β was under the detectable level in ELISA. Cooling of heat-exposed cells from 40°C to 37°C considerably down-regulated cellular proMMP-9 synthesis. Furthermore, proMMP-9 level was greatly reduced in cells treated at lower temperatures, 20°C and 5°C. These fi ndings support our hypothesis that hyperthermia in the horse tendon induces tendinocytes to synthesize pro-infl ammatory cytokines and that the synthesis of these cytokines results in the up-regulation of gelatinases.Tendon injury, especially in the superfi cial digital flexor tendon (SDFT), has proved to be a major problem for racehorses. Recent studies have shown that about 10-30% of racehorses suffer from tendonitis (29). Although the etiology of tendonitis has been discussed in many reports (2,19,25), there is little information on the mechanism of tendon degradation. Pro-infl ammatory cytokines, such as tumor necrosis factor (TNF) α and interleukin (IL)-1β, and gelatinases in matrix metalloproteinases (MMPs) are involved in degradation of connective tissues (30,33,34,40), and these pro-infl ammatory cytokines and gelatinases are synthesized in tendinocytes (15,18,33). Additionally, TNF α and IL-1β are known to be initiators of the synthesis of gelatinases and of the synthesis and release of cytokines (3,7,8,10,24,28).

Comparative study of the properties of tendinocytes derived from three different sites in the equine superficial digital flexor tendon

“…The temperature in the MTJ is likely to increase easily compared with the temperature in other sites due to its wide movable region, and heat induced-gelatinization would therefore progress more rapidly in the MTJ than in other sites. In support of this speculation, our previous study revealed that a hyperthermia environment accelerated MMP-9 synthesis (18). Tendinocytes might adjust to the environment surrounding the tendon.…”

“…However, in culture medium samples, a gelatin zymogram showed significant activity for pro-MMP-9 and very weak activity for proMMP-2, and the activated forms of both MMP-2 and -9 were hardly detectable (18,19). Moreover, proMMP-9 showed positive immunohistochemical reaction, but proMMP-2 reaction was very weak in tendinocytes derived from the mM site, and proMMP-2 synthesis by tendinocytes did not change regardless of TNFα treatment (18). In the present study, we could not detect proMMP-2 in the gelatin zymogram in any tendinocytes derived from the three different sites (Fig.…”

“…To evaluate the cellular response to TNFα, some cultured tendinocytes were incubated with 10 ng/mL purified equine TNFα (kindly provided by Dr. Rikio Kirisawa, Rakuno Gakuen University, Japan). The value was optimized prior to experiments (18,19).…”

“…Therefore, TNFα has a stimulatory effect on production of proMMP-13 in cultured tendinocytes. Proinflammatory cytokines, such as IL-1β and TNFα, stimulate synthesis of MMPs in various types of cells (12,18,24,42). In a normal tendon, restricted MMP-13 expression was observed in some fibroblastic cells and vascular component in the endotenon, suggesting the participation of MMP-13 in maintenance of homeostasis in matrix turnover in a tendon.…”

“…We speculated that morphological structure corresponds to biomechanical function in some sites of the SDFT. We have recently succeeded in establishing a method for isolating tendinocytes from equine tendons, and cultivation of isolated tendinocytes has provided much information on the properties of these cells types (16,18). In this study, we examined and compared the properties of tendinocytes obtained from MTJ, mM and OTJ sites of the SDFT, including morphology, proliferation, and production of collagen and MMPs.…”