Abstract
N-formyl-Met-Leu-Phe (fMLF) is a model PAMP/DAMP driving human PMN to sites of injury/infection utilizing the GPCR, FPR1. We examined a microtiter plate format for measurement of FPR1 phosphorylation in adherent PMN at high densities and found that a new phosphosensitive FPR1 fragment, 25K-FPR1, accumulates in SDS-PAGE extracts. 25K-FPR1 is fully inhibited by diisopropylfluorophosphate PMN pretreatment but is not physiologic, as its formation failed to be significantly perturbed by ATP depletion, time and temperature of adherence, or adherence mechanism. 25K-FPR1 was minimized by extracting fMLF-exposed PMN in lithium dodecylsulfate at 4°C prior to reduction/alkylation. After exposure of adherent PMN to a 5 log range of PAF before or after fMLF, unlike in suspension PMN, no inhibition of fMLF-induced FPR1 phosphorylation was observed. However, PAF induced the release of 40% of PMN lactate dehydrogenase, implying significant cell lysis. We infer that PAF-induced inhibition of fMLF-dependent FPR1 phosphorylation observed in suspension PMN does not occur in the unlysed adherent PMN. We speculate that although the conditions of the assay may induce PAF-stimulated necrosis, the cell densities on the plates may approach levels observed in inflamed tissues and provide for an explanation of PAF's divergent effects on FPR1 phosphorylation as well as PMN function.
Highlights
Human polymorphonuclear leukocytes (PMN, neutrophils) are the first line of cellular defense in the blood and tissue [1]
We found that FPR1, in adherent PMN, was more sensitive to proteolysis than its suspension cell counterpart, producing a 25 kDa fragment, 25K-FPR1, containing both carboxyl-terminal tail epitopes recognized by the FPR-specific mAbs and showing sensitivity to fMLF activation of PMNs
We found that in suspension PMN after 100 nM platelet-activating factor (PAF) treatment, this value did not decrease with exposure of PMN to 1 μM fMLF [24]
Summary
Human polymorphonuclear leukocytes (PMN, neutrophils) are the first line of cellular defense in the blood and tissue [1] They are the host’s protection against microbial pathogens and intrinsically involved in general injury and inflammation, recognizing both pathogen-activating molecular patterns (PAMPS) and damage-activating molecular patterns (DAMPS) [2]. PMN are equipped to deploy their defensive functions with massive cellular accumulation at sites of inflammation upon activation by diverse stimuli, including bacterial and mitochondrial products, denatured proteins, inflammatory lipids, complement components, and certain cytokines [4] They actively produce superoxide and secrete preformed polypeptides, lipids, and other agents as well as synthesize new components that protect the host by killing and degrading invading pathogens [5]. PMN aid in regulating [8] and dampening such injury by responding to anti-inflammatory lipid mediators, such as lipoxins and resolvins [9], as well as antiinflammatory cytokines [10, 11]
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