Effects of semisynthetic pullulan sulfates on the release of inflammatory cytokines in vitro

An important step of an inflammatory response is the production and release of cytokines like tumor necrosis factor-alpha (TNF-α) or interleukin-6 (IL-6) by human polymorphonuclear neutrophils (PMN) and monocytes (MC). Therefore, the inhibition of that release represents a promising approach to treat inflammatory diseases. Heparin was shown to exhibit anti-inflammatory activities and to affect the release of cytokines [1]. However, due to its high anticoagulant activity, its clinical application in inflammatory diseases is limited [2].

To develop glucan sulfates with an improved activity profile compared to heparin, series of pullulan sulfates (PulS) were produced by sulfation of the linear α-1,4/1,6-glucan from the yeast Aureobasidium pullulans. In the present study, the structure-dependent effects of PulS on the cytokine release were investigated.

PMN and MC, isolated from blood of healthy volunteers, were activated with 0.1µg/ml LPS in presence or absence of the test compounds (conc. ranging from 0.005 to 50µg/ml). The conc. of released TNF-α and IL-6 were measured by ELISA.

PulS showed to reduce the release of TNF-α and IL-6 from PMN and MC, resp., stronger than unfractionated heparin (UFH). Already at 0.005µg/ml, inhibitory effects between 30 and 60% were observed. With increasing conc. the inhibition only slightly increased. Comparing PulS with a similar MW of 10 kDa, but degrees of sulfation (DS) ranging from 0.2 to 2.0, the inhibitory activity improved up to a DS=1.5. In the absence of LPS, PulS do not induce any significant cytokine release. Moreover, the cytokine detection by ELISA is not impaired by the test compounds.

In summary, semisynthetic PulS with reduced anticoagulant activity are superior to heparin in inhibiting the cytokine release of activated leukocytes, which is in line with their increased anti-inflammatory activity in vivo.

References: [1] Hochart, H. (2006) Br. J. Haematol. 133: 62–67 [2] Becker, M. et al. Thromb. Haemost. (2003) 89: 915–25