Stroma Cell-Derived Factor 1α Mediates Desensitization of Human Neutrophil Respiratory Burst in Synovial Fluid from Rheumatoid Arthritic Patients

Abstract

Classical chemoattractants such as fMLP or the complement factor C5a use G protein (Gi)-coupled receptors to stimulate both chemotaxis and production of reactive oxygen species (respiratory burst, RB) by polymorphonuclear leukocytes (PMN). The chemokine stroma cell-derived factor 1α (SDF1α) and its Gi-coupled receptor, CXCR4, regulate leukocyte trafficking and recruitment to the synovial fluid of rheumatoid arthritic patients (RA-SF). However, the role of SDF1α in the RB is unknown and was studied in this work in vitro with healthy PMN in the absence and presence of RA-SF. In healthy PMN, SDF1α failed to stimulate the RB, even though the p38 mitogen-activated protein kinase was activated to a similar level as in fMLP-stimulated PMN. In contrast, the SDF1α-mediated calcium transients and activation of phosphatidylinositol 3-kinase/Akt were partially deficient, while p44/42 mitogen-activated protein kinases were not activated. SDF1α actually desensitized weakly the fMLP-mediated RB of healthy PMN. This cross-inhibitory effect was amplified in PMN treated with RA-SF, providing a protection against the exacerbation of RB induced by C5a or fMLP. This SDF1α beneficial effect, which was prevented by the CXCR4 antagonist AMD3100, was associated with impairment of C5a- and fMLP-mediated early signaling events. Thus, although SDF1α promotes leukocyte emigration into rheumatoid synovium, our data suggest it cross-desensitizes the production of oxidant by primed PMN, a property that may be beneficial in the context of arthritis.

Protein Kinase B (AKT) Mediates Phospholipase D Activation via ERK1/2 and Promotes Respiratory Burst Parameters in Formylpeptide-stimulated Neutrophil-like HL-60 Cells

“…A role of AKT in regulating RB was also suggested by the use of an AKT peptide antagonist and by co-immunoprecipitation studies of AKT with p47 phox in vivo (18). However, a number of observations suggest that activated AKT by itself is not sufficient to activate NADPH oxidase of intact cells directly: (i) AKT phosphorylation is stimulated by neutrophil stimuli that were unable to stimulate RB such as GM-CSF (27) or SDF1␣ (20); (ii) in dHL-60 cells stimulated by PMA, AKT was strongly phosphorylated ( Fig. 1) (33,34) and by PKA for Ser 328 (33).…”

“…Treatment of dHL-60 cells with fMLP induced a rapid and transient phosphorylation of AKT (Fig. 1B) peaking at approximately 1 min of cell stimulation and then returning to basal values within 5 min, as in primary human neutrophils (20). Cell treatment with 0.5-10 M AKTib1/2 reduced basal phosphorylation of AKT and abrogated fMLP-induced AKT phosphorylation (Fig.…”

“…RB of d-HL-60 cells was monitored continuously using the cytochrome c reduction assay (20). Aliquots of 2 ϫ 10 6 cells/ml of HBSS were stimulated under conditions used for the PLD assay.…”

“…Blocking AKT Stimulation Prevents fMLP-but Not PMA-mediated RB of dHL-60 Cells-AKT has been proposed to stimulate neutrophil RB due to its ability to phosphorylate the NADPH oxidase component p47 phox (26), although controversial studies have been reported (20,27). We took advantage of the potent AKTib1/2-blocking effects to examine further the contribution of AKT in RB.…”

“…2, C and D) confirmed that ERK phosphorylation was strongly inhibited by low concentrations of AKTib1/2 (IC 50 of 0.5 M). Taking advantage of the selective inhibitory effect of AKTib1/2, we examined whether another MAPK stimulated by fMLP, p38 MAPK (20), was regulated by AKT. In contrast to ERK, the rapid and transient phosphorylation of p38 MAPK induced by fMLP was not altered by AKTib1/2 (Fig.…”