Ahmad M. Kamal

A novel calcium-dependent proapoptotic effect of annexin 1 on human neutrophils

The glucocorticoid‐inducible protein annexin (ANXA) 1 is an anti‐inflammatory mediator that down‐regulates the host response. Endogenously, ANXA1 is released in large amounts from adherent polymorphonuclear neutrophils (PMN) and binds to their cell surface to inhibit their extravasation into inflamed tissues. The present study determined the effects of exogenous ANXA1 on several functions of human PMN in vitro. Addition of 0.1–1 µM human recombinant ANXA1 to the PMN provoked rapid and transient changes in intracellular Ca2+ concentrations that were blocked by the Ca2+ channel inhibitor SKF‐96365. Although ANXA1 did not affect oxidant production and only minimally affected PMN chemotactic properties, the ANXA1‐promoted Ca2+ influx was associated with two important functional effects: shedding of L‐selectin and acceleration of PMN apoptosis. The latter effect was confirmed using three distinct technical procedures, namely, cell cycle, Hoechst staining, and ANXA5 binding assay. ANXA1‐induced PMN apoptosis was insensitive to inhibitors of L‐selectin shedding, whereas it appeared to be associated with dephosphorylation of the proapoptotic intracellular mediator BAD. In conclusion, exogenous ANXA1 displayed selective actions on human PMN. We propose that the new proapoptotic effect reported here may be part of the spectrum of ANXA1‐mediated events involved in the resolution of acute inflammation.

An overview of the effects of annexin 1 on cells involved in the inflammatory process.

The concept of anti-inflammation is currently evolving with the definition of several endogenous inhibitory circuits that are important in the control of the host inflammatory response. Here we focus on one of these pathways, the annexin 1 (ANXA1) system. Originally identified as a 37 kDa glucocorticoid-inducible protein, ANXA1 has emerged over the last decade as an important endogenous modulator of inflammation. We review the pharmacological effects of ANXA1 on cell types involved in inflammation, from blood-borne leukocytes to resident cells. This review reveals that there is scope for more research, since most of the studies have so far focused on the effects of the protein and its peptido-mimetics on neutrophil recruitment and activation. However, many other cells central to inflammation, e.g. endothelial cells or mast cells, also express ANXA1: it is foreseen that a better definition of the role(s) of the endogenous protein in these cells will open the way to further pharmacological studies. We propose that a more systematic analysis of ANXA1 physio-pharmacology in cells involved in the host inflammatory reaction could aid in the design of novel anti-inflammatory therapeutics based on this endogenous mediator.

Formyl-peptide receptor is not involved in the protection afforded by annexin 1 in murine acute myocardial infarct

Recent interest in the annexin 1 field has come from the notion that specific G‐protein‐coupled receptors, members of the formyl‐peptide receptor (FPR) family, appear to mediate the anti‐ inflammatory actions of this endogenous mediator. Administration of the annexin 1 N‐terminal derived peptide Ac2‐26 to mice after 25 min ischemia significantly attenuated the extent of acute myocardial injury as assessed 60 min postreperfusion. Evident at the dose of 1 mg/kg (~9 nmol per animal), peptide Ac2‐26 cardioprotection was intact in FPR null mice. Similarly, peptide Ac2‐26 inhibition of specific markers of heart injury (specifically myeloperoxidase activity, CXC chemokine KC contents, and endogenous annexin 1 protein expression) was virtually identical in heart samples collected from wild‐type and FPR null mice. Mouse myocardium expressed the mRNA for FPR and the structurally related lipoxin A4 receptor, termed ALX; thus, comparable equimolar doses of two ALX agonists (W peptide and a stable lipoxin A4 analog) exerted cardioprotection in wild‐type and FPR null mice to an equal extent. Curiously, marked (>95%) blood neutropenia produced by an anti‐mouse neutrophil serum did not modify the extent of acute heart injury, whereas it prevented the protection afforded by peptide Ac2‐26. Thus, this study sheds light on the receptor mechanism(s) mediating annexin 1‐induced cardioprotection and shows a pivotal role for ALX and circulating neutrophil, whereas it excludes any functional involvement of mouse FPR. These mechanistic data can help in developing novel therapeutics for acute cardioprotection.

Antiflammin-2 Activates the Human Formyl- Peptide Receptor Like 1

The anti-inflammatory actions of the nonapeptide antiflammin-2, identified by homology with uteroglobin and annexin-A1 sequences, have been described in some detail, yet its mechanisms of action remain elusive. Since recent data indicate an involvement of the formyl peptide receptor (FPR)-like 1 (or FPRL-1) in the effects of annexin-A1, we have tested here the effect of antiflammin-2 with respect to this receptor family. Using HEK-293 cells expressing either human FPR and FPRL-1, and an annexin-A1 peptide as tracer ([125I-Tyr]-Ac2-26), we found that antiflammin-2 competed for binding only at FPRL-1, and not FPR, with an approximate EC50 of 1 μM. In line with data produced for the full-length protein, genuine receptor activation by antiflammin-2 was confirmed by rapid phosphorylation of extracellular-regulated kinase 1 and 2. Finally, study of the neutrophil interaction with activated endothelium under flow demonstrated an inhibitory effect of antiflammin-2, thus providing functional support to a role for the antiflammin-2/FPRL-1 anti-inflammatory axis.

Downstream Gene Activation of the Receptor ALX by the Agonist Annexin A1

Background Our understanding of pro-resolution factors in determining the outcome of inflammation has recently gained ground, yet not many studies have investigated whether specific genes or patterns of genes, are modified by these mediators. Here, we have focussed on the glucocorticoid modulated pro-resolution factor annexin A1 (AnxA1), studying if its interaction with the ALX receptor would affect downstream genomic targets. Methodology/Principal Findings Using microarray technology in ALX transfected HEK293 cells, we discovered an over-lapping, yet distinct gene activation profile for AnxA1 compared to its N-terminal mimetic peptide Ac2-26, which may be suggestive of unique downstream inflammatory outcomes for each substance. When the up-regulated genes were explored, consistently induced was the sphingosine phosphate phosphatase-2 gene (SGPP2), involved in regulation of the sphingosine 1 phosphate chemotactic system. Up-regulation of this gene, as well as JAG1 (and down-regulation of JAM3), was confirmed using real time PCR both with transfected HEK293 cells and human peripheral blood leukocytes. Furthermore, lymph nodes taken from AnxA1null mice displayed lower SGPP2 gene activity. Finally, connectivity map analysis for AnxA1 and peptide Ac2-26 indicated striking similarities with known anti-inflammatory therapeutics, glucocorticoids and aspirin-like compounds, as well as with histone deacetylase inhibitors. Conclusion/Significance We believe these new data raise the profile of AnxA1 from being solely a short-term anti-inflammatory factor, to being a ‘trigger’ of the endogenous pro-resolution arsenal.