Intracellular signaling in diabetic neutrophils (PMN)

Cytosolic signaling, i.e. changes in calcium concentration (Δ[Ca²⁺]_i) and pH (ΔpH_i), leads to activation of PMN.  Abnormalities in PMN signaling in people with diabetes mellitus (DM) have been reported to be related to modulation of bactericidal functions.  Objective: The objective of this study was to monitor PMN resting Ca²⁺ ([Ca²⁺]₀), Δ[Ca²⁺]_i and ΔpH_i in response to stimulation with f-Met-Leu-Phe (fMLF) or high-valency-immune-complexes (IC).  Methods:  PMN from peripheral blood of DM subjects and age, gender matched controls (C) were prepared with the fluorescent indicators Indo-1 for [Ca²⁺]_i, BCECF for pH_i, washed, incubated in de-aerated KRP (PBS +0.9mM Ca²⁺ & 1.5mM Mg²⁺) at 37°C for 2min, then stimulated with saturating doses [10^-7M] of fMLF or [120µg/ml] of IC.  Fluorescence change was measured on a Hitachi spectrofluorimeter.  Blood glucose and glycated hemoglobin (HbA_1c) levels were determined.  Results:  [Ca²⁺]₀ of DM PMN was 10% higher than C (ANOVA, p<0.01).  After stimulation with fMLF or IC, Δ[Ca²⁺]_i in DM PMN was reduced »10% compared to C.  The simultaneous cytosolic acidification in DM PMN was also weaker.  Conclusions:  These findings suggest that lower cytosolic Ca²⁺ and pH_i signals are associated with poor glycemic control suggesting a link between hyperglycemia and altered cytosolic signaling in subjects with DM.

Support: Alexander von Humboldt Foundation, NIH grants DE15566, DK31056, HL76463