The effects of a novel resuscitation strategy combining pentoxifylline and hypertonic saline on neutrophil MAPK signaling

Background. The combination of hypertonic saline (HS) and pentoxifylline (PTX) has been shown to synergistically downregulate neutrophil oxidative burst in vitro. We investigated the effects of HS/PTX on human neutrophil mitogen-activated protein kinase (MAPK) signaling and the role of Protein kinase A (PKA) in this process. Methods. Isolated neutrophils were treated with PTX (2 mmol/L), HS10 (10 mmol/L above isotonicity), and HS40 (40 mmol/L above isotonicity) alone or in combination for determination of intracellular cyclic adenosine monophosphate (cAMP) concentrations. Human neutrophils were stimulated with f-methionyl-leucyl-phenylalanine (fMLP) (1 mu mol/L) before the treatments above in both the presence and the absence of PKA inhibition for Western blot analysis of MAPK p38 and extracellular signal-related kinase 1/2 (ERK 1/2) phosphorylation. Results. Concomitant exposure to HS/PTX results in an additive increase in intracellular cAMP. fMLP-induced ERK 1/2 phosphorylation was synergistically attenuated by HS/PTX. Both PTX and HS reduced p38MAPK phosphorylation. No additive effect was observed with combined treatment. Although PKA inhibition abrogated the effects of PTX, HS retained some capacity to attenuate MAPK phosphorylation. Conclusion. HS/PTX is more effective in attenuating neutrophil ERK signaling than either component alone, whereas both components alone or in combination produced comparable results with p38MAPK. Although PTX functions primarily through PKA activation, HS may suppress neutrophils through a partially PKA-independent mechanism.