1. FITC Conjugation Markedly Enhances Hepatic Clearance of N-Formyl Peptides
Baldur Sveinbjørnsson, Bård Smedsrød, Kjetil Elvevold, Igor Snapkov, Cristina Ionica Øie PLoS One . 2016 Aug 5;11(8):e0160602. doi: 10.1371/journal.pone.0160602.
In both septic and aseptic inflammation, N-formyl peptides may enter the circulation and induce a systemic inflammatory response syndrome similar to that observed during septic shock. The inflammatory response is brought about by the binding of N-formyl peptide to formyl peptide receptors (FPRs), specific signaling receptors expressed on myeloid as well as non-myeloid cells involved in the inflammatory process. N-formyl peptides conjugated with fluorochromes, such as fluorescein isothiocyanate (FITC) are increasingly experimentally used to identify tissues involved in inflammation. Hypothesizing that the process of FITC-conjugation may transfer formyl peptide to a ligand that is efficiently cleared from the circulation by the natural powerful hepatic scavenging regime we studied the biodistribution of intravenously administered FITC-fNLPNTL (Fluorescein-isothiocyanate- N-Formyl-Nle-Leu-Phe-Nle-Tyr-Lys) in mice. Our findings can be summarized as follows: i) In contrast to unconjugated fNLPNTL, FITC-fNLPNTL was rapidly taken up in the liver; ii) Mouse and human liver sinusoidal endothelial cells (LSECs) and hepatocytes express formyl peptide receptor 1 (FRP1) on both mRNA (PCR) and protein (Western blot) levels; iii) Immunohistochemistry showed that mouse and human liver sections expressed FRP1 in LSECs and hepatocytes; and iv) Uptake of FITC-fNLPNTL could be largely blocked in mouse and human hepatocytes by surplus-unconjugated fNLPNTL, thereby suggesting that the hepatocytes in both species recognized FITC-fNLPNTL and fNLPNTL as indistinguishable ligands. This was in contrast to the mouse and human LSECs, in which the uptake of FITC-fNLPNTL was mediated by both FRP1 and a scavenger receptor, specifically expressed on LSECs. Based on these results we conclude that a significant proportion of FITC-fNLPNTL is taken up in LSECs via a scavenger receptor naturally expressed in these cells. This calls for great caution when using FITC-fNLPNTL and other chromogen-conjugated formyl peptides as a probe to identify cells in a liver engaged in inflammation. Moreover, our finding emphasizes the role of the liver as an important neutralizer of otherwise strong inflammatory signals such as formyl peptides.
2. Covalent affinity labeling of the formyl peptide chemotactic receptor
P Cuatrecasas, J Niedel, J Davis J Biol Chem . 1980 Aug 10;255(15):7063-6.
The formyl peptide chemotactic receptor present on human neutrophil membranes has been covalently affinity-labeled using three different techniques. N-Formyl-Nle-Leu-Phe-Nle-125I-Tyr-Lys was cross-linked to the receptor with dimethyl suberimidate, N-formyl-Nle-Leu-Phe-Nle-125I-Tyr-Lys-Nepsilon-4-azido-2-nitrophenyl was cross-linked by photoactivation, and N-formyl-Nle-Leu-Phe-Nle-125I-Tyr-Lys-Nepsilon-bromoacetyl reacted spontaneously with the receptor. With each method, a polypeptide which migrated as a broad band on sodium dodecyl sulfate-polyacrylamide electrophoresis, with an apparent molecular weight between 55,000 and 70,000, was specifically labeled. A dose-response curve for inhibition of covalent cross-linking of the formyl peptide derivatives by unlabeled N-formyl-Nle-Leu-Phe-Nle-Tyr-Lys correlated closely with a dose-response curve for inhibition of binding of the same formyl peptide derivatives to the receptor. The nonformylated analog, Nle-Leu-Phe-Nle-Tyr-Lys, did not inhibit binding or covalent cross-linking.
3. Effects of monomethylfumarate on human granulocytes
B Thio, T P Zomerdijk, A C Bezemer, P H Nibbering, R L Beijersbergen, R van Furth J Invest Dermatol . 1993 Jul;101(1):37-42. doi: 10.1111/1523-1747.ep12358715.
Monomethylfumarate (MMF) is the most active metabolite of the new antipsoriasis drug Fumaderm. Because granulocytes play an important role in the pathophysiology of psoriasis, the effects of this drug on the functional activities of these cells were investigated. MMF stimulated polarization and elastase release, and enhanced the intracellular killing of bacteria by granulocytes. This compound suppressed the formyl-Met-Nle-Phe (FMLP)-stimulated respiratory burst in these cells. MMF and dimethylfumarate but not its stereoisomer dimethylmaleate, fumaric acid, or dimethylmalate stimulated polarization of and elastase release by granulocytes, indicating that methylated fumarate derivatives interact with granulocytes in a specific fashion. MMF did not affect the binding of formyl-Nle-Leu-Phe-Nle-Tyr-Lys-fluorescein isothiocyanate to the FMLP receptor on granulocytes. This compound induced an increase in the intracellular Ca++ ([Ca++]i) and cyclic adenosine monophsphate concentration. The agonistic effects of MMF on granulocytes are thought to be mediated by the rise in the [Ca++]i and the antagonistic effects by the increase in the cyclic adenosine monophosphate concentration. These effects of MMF on granulocytes may in part explain the beneficial action of methylated fumarate derivatives on psoriatic skin lesions.
