Abstract
Interleukin (IL) 1 alpha is synthesized as a 33-kDa precursor that is enzymatically cleaved to the 15-17-kDa forms that are found in the culture supernatants of activated macrophages. We have explored the possibility that calcium might enhance IL-1 processing and secretion via the stimulation of a calcium-dependent protease. We have found that lysates prepared from human peripheral blood monocytes, the human histiocytic lymphoma cell line U937, and the murine macrophage cell line P388D1 contain a calcium-dependent IL-1 alpha processing activity that cleaves the IL-1 alpha precursor to its mature form. Although NIH 3T3 mouse fibroblast cell lysates also contain IL-1 processing activity, lysates from the murine thymoma EL-4, the human epidermoid cell line HEp-2, and the human foreskin fibroblast line FS-4 lack this activity. IL-1 processing activity is inhibited by leupeptin and exhibits a molecular mass of 80-110 kDa. The processing activity is also inhibited by a monoclonal antibody directed against calpain type I. These results indicate that the processing of the IL-1 alpha precursor is mediated, at least in part, by a member of the calpain family of proteases. Mixing experiments revealed that lysates from EL-4 or HEp-2 cells contain an inhibitor(s) of the calpain-like protease in macrophage extracts. It is, therefore, likely that many non-macrophage cell types are unable to process the IL-1 alpha precursor because the calpain present in these cells is only weakly active due to the presence of a specific inhibitor(s) such as calpastatin.
Highlights
Cells-The murine macrophage cell line P388D1, the murine thymoma cell line EL-4, and the human histiocytic lymphoma cell line immune and inflammatory responses including antigen and U937 were grown insuspensionculture at 37 “CinRPMI 1640 mitogen-dependent lymphocyte activation [1, 2], fever [3], (GIBCO) supplemented wit1h0% fetal bovine serum (KCBiologicals) and the synthesis of several acute-phase response proteins and 50 +g/ml gentamicinsulfate
Cloning studies revealed the existence of two forms of human foreskin fibroblasctell line FS-4, NIH 3T3 murifniberoblasts, human and murine IL-1, termed IL-laIL-a1nPd, that possess and the human epidermoid carcinoma line HEp-2 were grown as monolayers in RPMI1640 supplemented with10% fetal bovine serum and gentamicin sulfate
The 1.1-kilobase pIL-la cDNA was excised and inserted into the HincII siteof pGEM-3z (Promega) andamplified in Escherichia coli using standardmethods[13].Purifiedplasmid was linearized with Hind111 or EcoRI and transcribedusing SP6 or T7 polym
Summary
IL, interleukin; EGTA, [ethylene- erasein a n in vitro transcription system (Promega). The mRNAs bis(oxyethylenenitri1o)ltetraacetic acid; E-64, trans-epoxysuccinyl-L- were processed according to the manufacturer’s instructionsI.n vitro leucylamido-(4-guanidino)butane;CANP, calcium-activated neutral translations were performed using micrococcal nuclease-treated rabproteasHe;EPES, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic bit reticulocyte extracts (PromegaBiotec) supplemented with invitro acid; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electro- synthesized mRNA and25 +Ci/ml [”S]methionine. Preparation of Cell-free Extracts-Cell-free extracts were prepared by hypotonic lysis of cells. Adherent cells were washed twice with from the membrane using an autoradiograph of the membrane as a phosphate-buffered saline (10 mM phosphate, pH 7.4, 150 mM NaCI) guide. Amino acid sequenceanalysis was performed using anApplied and once with lysis buffer
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
