Generation of interleukin‐8 by plasmin from AVLPR‐interleukin‐8, the human fibroblast‐derived neutrophil chemotactic factor

Abstract

Phtsmin mai,dy cle;tved the ArlI~,$eP bond o1' ArlpVaI.Leu.Pro.Arll.inletleukin.8 (AVLPR-IL-a} produced by human derm.I Itbroblast=t, which r¢=ulted in the conversion orAVLPR.IL.8 to IL.~t and the in,clive pentapeptide, though, minor cleavall~ of AVLPR-IL.It by phtsmin at Ly~':,Ght'* bond occurred. MATERIALS AND METHODS Purtfication of FDNCFHuman dermal fibroblasts (SF-TY cell line) were maintained in Dulbecco's modified Eagle's medium supplemented with 5°7o fetal calf sert~,m, 25 mM Hepes, penicillin (0, I mg/ml) and streptomycin (O. 1 mg/ml) until they reached confluence. The cells were cultured in serum-free culture medium supplemented with tO "m M recombinant human IL-I# and 0.1o70 bovine serum albumin. After culture for 2 days, cell.free conditioned medium was obtained by centrifugation at 1600× g for 20 rain. The chemoattractant in the conditioned medium Abbreviations; AVLPR-IL-8, Ala-Val-Leu-Pro-Arg-interleukin-8; FDNCF fibroblast-derived neutrophil chemot~ctic factor; RP. HPLC, reverse-phase high performance liquid chromatography; SDS-PAGE, sodium dodccyl sulfate polyacrylamide gel electrophoresJs.Correspondence address: H. Nakagawa, Faculty of Pharmaceutical Sciences, Toyama Medical and Pharmaceutical University, Sugitani, Toyarna 930-01, Japan, was purified essentially ns described previously [11]; the sample was chromatoliraphedsequential/y on CM-Seph~dex, Sephadex O-?$ and RP-HPLC, Tree/men/of FDNCF =+'ith plasminFDNCF (40 j+g) in a final volume of 0,2 ml ¢0f 50 mM Tris.HCI buffer (pH 8.0) was incubated with 0,gpg of plasmin (purified from human plasma; Sigma, MO. USA) at 37*C for 30 rain, and the rent. lion was stopped by the addition of leupeptin (final 1 raM), The reae. lion mixture was concentrated by a Speed-Vac centrifuge, dissolved in 6 M 8uanidine solution and loaded onto a C.18 reverse-phase col. umn (0.45 x l$cm; ODS.120T, Tosoh Co, Tokyo, Japan).Chemotaetic factors were ehtted with a linear co~centration gradient of aeetonitrile from 0% to 50.4% in 0.05% trifluoroacetic acid at z~ flow rate of 0.8 ml/min, Neutrophil chemotaxls of plasmin.treated FDNCF was performed in vitro by using rat and human neutrophils as described previously [121, As an index of ehemotaxis, the number of neutrophils migrated into the lower chamber was expressed as percentage (migration rate) of that of neutrophils applied in the upper chamber, $DS-PAGE was carried out on a slab gel of a discontinuous 9,6%/16,507o acrylamide in tricine buffer system [13]. NH2.terminal atnino acid sequencing analysisNH,-terminal amino acid sequence determination was performed by automated Edman degradation on a gas-plmse protein sequencer (model 470A, Applied Biosystems, CA, USA) e¢~uipped with a PTH analyzer (model 120A HPLC system). RESULTS AND DISCUSSIONHuman dermal fibroblasts stimulated with 10 -t° M IL-1 mainly produced FDNCF, which is the NH2-termina] extended 77-residue variant of IL-8 (AVLPR-IL-8). The homogeneity of the purified FDNCF was confirmed by SDS-PAGE, its NH2-terminal amino acid sequence and its COOHterminal amino...

Citrullination of CXCL10 and CXCL11 by peptidylarginine deiminase: a naturally occurring posttranslational modification of chemokines and new dimension of immunoregulation

“…For example, the conversion of CXCL8(1-77) into CXCL8(6-77) by plasmin or thrombin potentiates its biologic activity, facilitating a positive feedback loop. 37,38 Moreover, posttranslational modifications seem to complicate the pattern of apparent redundancy in chemokinereceptor interactions. Indeed, after processing by DPP-IV/CD26, the CC chemokine CCL5/RANTES discloses an enhanced receptor interaction with CCR5, parallel though with a decreased affinity for CCR1 and CCR3.…”

Protein Chip Discovery of Secreted Proteins Regulated by the Phosphatidylinositol 3-Kinase Pathway in Ovarian Cancer Cell Lines

“…SELDI-TOF MS peak intensity at m/z f7,860 was plotted against CXCL1 concentration measured by RIA in duplicate media from all five cell lines subjected to four treatments at four time points (n = 160). and thrombin (21,22) and that the longer form is less potent at elastase release and inducing neutrophil chemotaxis (23). It is not known if there is a difference between the two forms in their proliferative and tumorigenic properties.…”

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“…Thrombin and plasmin were reported to truncate CXCL8 by removing five NH 2 -terminal amino acids to generate a more active CXCL8 isoform, previously isolated as an important CXCL8 form from both fibroblasts and endothelial cells [29,[36][37][38]. In vitro, the membrane-bound or soluble protease DPP IV or CD26 rapidly truncates CXCL10 (4 min half-life at a physiological CD26 concentration) [22].…”