_:b539158769 "2"^^ . _:b539158768 "2"^^ . . _:b539158901 . _:b539158771 "2"^^ . _:b539158787 . _:b539158768 . _:b539158770 "2"^^ . _:b539158884 . . _:b539158763 . _:b539158749 . _:b539158781 "2"^^ . _:b539158780 "2"^^ . . _:b27063100 "In the case of the precursor forms of IL-1\u03B1 and IL-33, N-terminal pro-sequence has another function, binding to nucleic DNA and therefore participating in transcription (3, >>5<<)." . _:b539158854 . . . _:b539158891 . _:b539158783 "2"^^ . . _:b539158887 . _:b539158782 "2"^^ . _:b539158777 "2"^^ . _:b539158743 . _:b539158776 "2"^^ . . _:b27063070 "However, in another study (>>4<<) keratinocytes exposed to prolonged hypoxia in vitro released cellular contents containing IL-1\u03B1 with a molecular weight of 30,000\u2009Da consistent with the precursor form." . _:b539158779 "2"^^ . . . _:b539158745 . _:b539158778 "2"^^ . _:b539158789 "2"^^ . _:b539158761 . . _:b539158788 "2"^^ . . . _:b539158882 . _:b539158735 . _:b539158791 "2"^^ . _:b539158790 "2"^^ . _:b539158785 "2"^^ . _:b27063109 . _:b27063109 . _:b27063062 . _:b539158849 . _:b27063079 "The human mast cell responses to IL-33 were measured in the HMC-1 cell line as described previously (6, >>19<<). After 24\u2009h of incubation with mature or precursor IL-33, IL-8 was measured in the supernatants (R&D Systems)." . _:b539158784 "2"^^ . . _:b27063087 "IL-33, another member of IL-1 family, acts as an alarmin (>>26<<\u201331). Precursor and mature forms of recombinant IL-33 were added to A549 cells and PBMCs." . _:b539158787 "2"^^ . _:b539158786 "2"^^ . _:b27063067 . . _:b539158789 . _:b27063054 . _:b539158797 "2"^^ . _:b539158788 . _:b539158778 . _:b27063055 . . _:b539158796 "2"^^ . _:b539158791 . _:b539158808 . . _:b539158799 "2"^^ . _:b539158790 . . . _:b539158701 . . . _:b539158798 "2"^^ . . _:b539158785 . . . . _:b27063068 . _:b539158793 "2"^^ . _:b539158784 . _:b539158792 "2"^^ . _:b27063069 . . _:b539158787 . . _:b27063054 "introduction" . . _:b27063073 "Since IL-1, either IL-1\u03B1 or IL-1\u03B2, promotes IL-17 production by T cells, alarmin-mediated immune responses may be an important endogenous activator of pathogenic T cells in autoimmune diseases (17, >>18<<)." . _:b27063060 . _:b539158795 "2"^^ . _:b27063112 "In other studies, IL-33 exhibits a weak activity as a precursor compared to mature IL-33 (6, >>19<<, 40)." . _:b539158786 . _:b539158809 . . _:b27063061 . . _:b539158794 "2"^^ . _:b539158797 . _:b27063062 . _:b539158805 "2"^^ . . _:b539158796 . . . _:b539158869 . _:b539158789 . . . _:b27063063 . _:b539158804 "2"^^ . _:b539158799 . _:b27063056 . _:b539158807 "2"^^ . _:b539158798 . _:b27063080 "results" . . _:b27063057 . _:b539158806 "2"^^ . _:b539158793 . _:b27063058 . _:b539158801 "2"^^ . _:b27063059 . . _:b539158792 . . _:b27063059 . _:b539158791 . _:b539158800 "2"^^ . . _:b539158795 . . _:b27063096 "Nevertheless, in vivo the IL-1\u03B2 precursor can be processed extracellularly by proteinase-3 and elastase into an active cytokine (9, >>35<<). There is no caspase-1 cleavage site in the IL-1\u03B1 precursor; cleavage is by a membrane associated calpain results in a 17,000\u2009Da peptide. The N-terminus of the calpain cleavage product is assumed to be the same as that the IL-1\u03B1 purified" . _:b27063068 . _:b539158803 "2"^^ . _:b539158794 . . _:b27063069 . . _:b27063054 . _:b539158802 "2"^^ . _:b539158805 . . _:b27063070 . . _:b539158881 . _:b539158813 "2"^^ . _:b539158773 . . . _:b539158804 . . _:b539158795 . _:b27063093 . _:b27063071 . _:b539158812 "2"^^ . _:b539158807 . _:b27063064 . _:b539158815 "2"^^ . _:b27063055 "Among the 11 members of IL-1 family cytokines, 10 have no signal peptide, the exception being the IL-1 receptor antagonist (IL-1Ra) (>>1<<). The signal peptide in IL-1Ra allows the cell to release the antagonist as an active molecule without a requirement for processing of the IL-1Ra precursor whereas all other members of the IL-1 family require processing from an inactive" . _:b539158806 . . _:b27063065 . _:b539158814 "2"^^ . _:b539158801 . _:b539158909 . _:b27063066 . . _:b539158809 "2"^^ . _:b539158800 . . . _:b27063067 . _:b539158851 . _:b539158811 . _:b539158808 "2"^^ . _:b539158797 . _:b539158803 . _:b27063088 . _:b539158870 . . _:b539158837 . _:b27063076 . _:b539158811 "2"^^ . _:b27063090 . . _:b539158802 . _:b539158814 . _:b27063077 . _:b539158810 "2"^^ . _:b539158813 . . _:b539158769 . _:b27063080 . _:b27063078 . _:b539158800 . _:b539158821 "2"^^ . _:b539158801 . _:b539158896 . _:b539158812 . _:b27063079 . _:b539158810 . _:b539158820 "2"^^ . . _:b539158815 . _:b27063082 "The IL-1R2 is non-signaling decoy receptor, which dampens IL-1\u03B2 driven inflammation (>>21<<) and preferentially binds mature IL-1\u03B2 but not IL-1\u03B1 (22)." . . _:b27063072 . _:b27063074 . _:b27063108 "Indeed, it has been reported that intracellular IL-1\u03B1 precursor is bound to intracellular IL-1R2 and that this complex inhibits the activity of precursor IL-1\u03B1 (>>37<<). In those cells without constitutive IL-1R2, the IL-1\u03B1 precursor is active. One can conclude from that study that necrotic cells without constitutive IL-1R2 are likely to induce inflammation due to the activity of the IL-1\u03B1 precursor" . _:b539158823 "2"^^ . _:b539158758 . _:b539158722 . _:b539158814 . _:b27063073 . . _:b539158822 "2"^^ . _:b539158772 . _:b539158809 . . _:b27063074 . _:b539158817 "2"^^ . _:b539158808 . . _:b539158852 . . _:b27063075 . _:b539158816 "2"^^ . _:b539158811 . _:b27063086 "This is particularly an important issue since little is known of the binding of the IL-1\u03B1 precursor to the IL-R1 compared to the binding of mature IL-1\u03B1, IL-1Ra, and mature IL-1\u03B2 (>>25<<). The rationale for testing the ability of the IL-1Ra to block the responses provides indirect evidence that precursor IL-1\u03B1, mature IL-1\u03B1, and mature IL-1\u03B2 share the same receptor since IL-1Ra binds solely to IL-1R1. When we added IL-1Ra" . _:b27063066 . _:b27063056 "IL-1\u03B1 was the first cytokine shown to translocate to the nucleus and the pro-piece contains a nuclear localization sequence (LKKRRL), which binds to chromatin and acts as transcription factor (>>2<<\u20134). Although during apoptosis, the intracellular IL-1\u03B1 precursor binds tightly to chromatin (4), during necrosis, the precursor dissociates from the chromatin is released from the cell in the precursor form (4). The IL-33 precursor also" . _:b539158836 . _:b27063084 . _:b539158766 . _:b27063091 . _:b539158819 "2"^^ . _:b539158810 . _:b27063096 . _:b27063085 . _:b539158818 "2"^^ . _:b539158821 . . _:b27063086 . _:b539158829 "2"^^ . _:b539158820 . _:b27063087 . _:b539158828 "2"^^ . _:b539158823 . _:b27063064 . _:b27063080 . _:b539158831 "2"^^ . . _:b539158822 . _:b27063081 . _:b539158830 "2"^^ . _:b539158817 . _:b539158839 . _:b539158832 . _:b539158842 . _:b27063082 . _:b539158825 "2"^^ . . _:b539158816 . . _:b27063083 . _:b539158824 "2"^^ . _:b27063101 . _:b539158819 . _:b27063092 . . _:b539158827 "2"^^ . _:b539158818 . _:b27063093 . _:b27063113 . _:b539158726 . _:b27063100 . _:b539158826 "2"^^ . _:b539158829 . _:b27063094 . _:b539158837 "2"^^ . _:b539158828 . . . _:b27063095 . _:b539158836 "2"^^ . _:b539158827 . _:b539158831 . . . _:b27063088 . _:b27063103 . _:b539158839 "2"^^ . _:b539158806 . _:b539158830 . _:b27063112 . _:b27063089 . . _:b539158838 "2"^^ . . _:b539158825 . . _:b27063090 . _:b539158833 "2"^^ . _:b539158824 . _:b27063073 . _:b27063091 . . _:b539158832 "2"^^ . _:b539158827 . _:b539158816 . _:b27063100 . _:b539158835 "2"^^ . _:b539158826 . _:b27063101 . _:b539158834 "2"^^ . _:b539158837 . _:b539158729 . _:b27063102 . . . _:b539158845 "2"^^ . _:b539158836 . _:b27063103 . _:b539158844 "2"^^ . _:b539158898 . . _:b539158839 . . _:b27063110 . _:b27063096 . _:b539158847 "2"^^ . . _:b539158838 . . _:b27063097 . . _:b539158846 "2"^^ . _:b539158833 . . _:b27063098 . _:b539158841 "2"^^ . _:b539158832 . . _:b539158864 . _:b27063099 . _:b539158764 . _:b539158840 "2"^^ . _:b539158835 . _:b27063108 . . _:b539158843 "2"^^ . _:b539158834 . . _:b27063109 . _:b539158842 "2"^^ . . _:b539158845 . . _:b539158709 . . _:b27063110 . _:b539158853 "2"^^ . _:b539158844 . _:b27063111 . _:b539158852 "2"^^ . _:b27063065 . . _:b539158847 . _:b27063104 . . _:b539158855 "2"^^ . _:b539158846 . _:b27063105 . . _:b539158854 "2"^^ . _:b539158841 . . _:b27063106 . . _:b539158849 "2"^^ . _:b539158840 . _:b27063107 . _:b539158848 "2"^^ . . _:b539158843 . . _:b539158851 "2"^^ . _:b539158842 . . . _:b539158850 "2"^^ . _:b539158853 . _:b539158861 "2"^^ . _:b539158712 . _:b539158852 . _:b539158871 . _:b539158860 "2"^^ . . _:b539158855 . _:b27063112 . _:b539158863 "2"^^ . _:b539158818 . _:b539158854 . _:b27063113 . _:b539158862 "2"^^ . _:b539158849 . . _:b539158771 . _:b539158857 "2"^^ . _:b539158848 . . _:b539158710 . _:b539158856 "2"^^ . _:b539158855 . _:b539158851 . _:b27063076 "The N-termini for mature IL-1\u03B2 was at 117 (caspase-1 site), for mature IL-1\u03B1 was 115 and for IL-33 111 (>>19<<). Anti-human IL-1\u03B1 was a kind gift of XBiotech (Austin, TX, USA) and IL-1Ra (anakinra) was purchased from R&D Systems, Minneapolis, MN, USA." . _:b539158750 . . _:b539158822 . _:b539158859 "2"^^ . _:b539158850 . _:b539158885 . _:b27063077 "Human A549 cells were cultured as described previously (>>20<<). Recombinant cytokines were added in presence or absence of anti-IL-1\u03B1 or IL-1Ra as described above." . _:b539158858 "2"^^ . _:b539158861 . . _:b27063087 . _:b539158869 "2"^^ . _:b539158860 . _:b539158793 . . _:b539158868 "2"^^ . _:b539158863 . _:b539158894 . . _:b27063071 . _:b539158871 "2"^^ . _:b539158779 . _:b539158862 . _:b539158731 . _:b539158870 "2"^^ . _:b539158857 . _:b539158746 . . _:b539158865 "2"^^ . _:b539158856 . _:b539158864 "2"^^ . _:b27063092 "when the precursors for IL-1\u03B2 or IL-1\u03B1 or IL-33 are released from necrotic cells into a neutrophil-rich microenvironment, extracellular processing can take place generating one or more mature forms with different levels of activity (>>7<<\u20139). Indeed, the biological and receptor binding activities of 23 unique amino acid sequences of mature human IL-1\u03B1 (111\u2013270) were expressed in E. coli and compared with WT the activity of the mature molecule." . _:b539158859 . . _:b539158799 . _:b539158867 "2"^^ . _:b539158858 . _:b539158724 . _:b539158866 "2"^^ . _:b539158869 . . _:b539158877 "2"^^ . _:b539158868 . . . . _:b539158876 "2"^^ . _:b539158871 . _:b539158879 "2"^^ . . _:b539158870 . . _:b539158782 . _:b539158813 . _:b539158878 "2"^^ . _:b539158865 . _:b539158754 . . _:b539158873 "2"^^ . _:b539158864 . _:b539158872 "2"^^ . _:b539158867 . _:b27063095 . . _:b539158875 "2"^^ . _:b539158866 . . _:b27063092 . . _:b539158874 "2"^^ . _:b539158877 . _:b539158913 . _:b27063107 "It is known that some forms of tissue necrosis are associated with inflammation whereas others are not (>>37<<). Thus, one can assume that the activity of IL-1\u03B1 maybe under a control mechanism which is cell specific. Indeed, it has been reported that intracellular IL-1\u03B1 precursor is bound to intracellular IL-1R2 and that this complex inhibits the" . _:b539158885 "2"^^ . _:b539158876 . . _:b539158884 "2"^^ . . _:b539158879 . _:b539158887 "2"^^ . _:b539158878 . _:b539158886 "2"^^ . _:b539158873 . _:b539158881 "2"^^ . . _:b539158872 . "10.3389%2Ffimmu.2013.00391" . _:b27063106 "These apoptotic bodies are highly inflammatory in vivo and are due to IL-1\u03B1 and not IL-1\u03B2 (>>38<<). Another role for inflammation due to the IL-1\u03B1 precursor is in dying tumors. As tumors outgrow their vascular supply, necrosis takes place and here the source of the IL-1\u03B1 precursor is the tumor itself. Proof of this mechanism comes" . _:b539158798 . . . _:b539158775 . _:b539158880 "2"^^ . _:b539158860 . _:b539158875 . _:b539158861 . _:b539158719 . _:b539158862 . _:b539158863 . _:b539158883 "2"^^ . . _:b539158856 . _:b539158874 . _:b539158857 . _:b539158858 . _:b539158859 . _:b539158882 "2"^^ . _:b539158852 . _:b539158885 . _:b539158853 . . _:b539158854 . _:b539158855 . _:b539158893 "2"^^ . _:b539158848 . _:b539158884 . _:b539158849 . . _:b539158850 . _:b539158851 . _:b539158892 "2"^^ . _:b539158876 . _:b539158893 . _:b539158887 . _:b539158877 . _:b539158878 . _:b539158879 . _:b539158895 "2"^^ . _:b539158872 . _:b539158886 . _:b539158873 . _:b539158874 . _:b539158875 . _:b27063104 "Others have reported that mature IL-1\u03B1 is more active than the precursor using a complex assay for IL-1 that is IL-1-augementation of IL-2 production from EL4 cells (>>37<<). In that study, the investigators encountered significant difficulties with the activity of N-terminal his-tagged mature IL-1\u03B1 due to aggregation. To circumvent the problem, the precursor was cleaved with calpain and the comparison of" . _:b539158894 "2"^^ . _:b539158776 . _:b539158868 . _:b539158881 . _:b539158869 . _:b539158870 . _:b539158871 . _:b539158889 "2"^^ . _:b539158864 . _:b27063056 . _:b539158880 . _:b539158865 . _:b539158866 . . _:b539158867 . _:b539158888 "2"^^ . _:b539158892 . _:b539158883 . _:b539158893 . _:b539158894 . . _:b539158895 . _:b539158891 "2"^^ . _:b539158888 . _:b539158882 . . _:b539158889 . _:b539158890 . _:b539158891 . _:b539158890 "2"^^ . _:b539158717 . _:b539158884 . _:b539158893 . _:b539158885 . _:b539158886 . _:b539158887 . _:b539158901 "2"^^ . _:b539158880 . _:b539158892 . _:b539158881 . _:b539158882 . _:b539158883 . _:b539158900 "2"^^ . _:b539158770 . _:b539158908 . _:b539158895 . _:b539158725 . _:b539158909 . . _:b539158910 . _:b539158911 . _:b539158903 "2"^^ . . _:b539158904 . _:b539158894 . . _:b539158905 . _:b539158906 . _:b539158907 . _:b539158902 "2"^^ . _:b539158805 . _:b539158900 . _:b539158889 . _:b539158901 . _:b539158902 . . _:b539158713 . _:b539158897 "2"^^ . _:b539158903 . _:b539158896 . _:b539158888 . _:b539158897 . _:b539158748 . _:b539158898 . _:b539158889 . _:b539158715 . _:b539158899 . _:b539158896 "2"^^ . _:b539158796 . _:b539158891 . _:b539158797 . _:b539158798 . _:b539158799 . _:b539158899 "2"^^ . _:b539158792 . _:b539158890 . _:b539158793 . _:b539158794 . _:b539158795 . _:b539158898 "2"^^ . _:b539158788 . _:b539158901 . _:b539158789 . . _:b539158790 . _:b539158791 . _:b539158909 "2"^^ . _:b539158784 . _:b539158900 . . . _:b539158785 . _:b539158786 . _:b539158850 . _:b539158787 . _:b539158908 "2"^^ . _:b539158812 . _:b539158903 . _:b539158813 . _:b539158814 . _:b27063065 "In acute lung injury, the type II epithelial cells contain constitutive IL-1\u03B1 precursor and when released, mediates local inflammation, which is prevented by IL-1 receptor blockade (>>13<<, 14). In a mouse model of DAMP activity from necrotic tumor cells injected intraperitoneally, there is neutrophilic inflammation but also elevation of hepatic enzyme alanine aminotransferase (ALT) (15). The inflammation was observed in" . _:b539158815 . _:b539158911 "2"^^ . . _:b539158808 . _:b539158902 . _:b539158809 . _:b539158810 . _:b539158811 . _:b539158910 "2"^^ . _:b539158863 . _:b539158804 . _:b539158897 . _:b539158805 . _:b539158806 . _:b539158807 . _:b27063091 "In the case of IL-1, specific antibody neutralization has been used to distinguish IL-1\u03B1 from IL-1\u03B2 (15, >>16<<)." . _:b539158905 "2"^^ . . _:b539158800 . _:b539158896 . . _:b27063062 . _:b27063062 "The precursor forms for IL-1\u03B2 and IL-18 are inactive; processing to an active cytokine can take place intracellularly by caspase-1 but extracellularly by neutrophil-derived proteinase-3 (>>8<<\u201310)." . _:b539158801 . _:b539158802 . _:b539158803 . _:b539158904 "2"^^ . _:b539158828 . _:b539158899 . _:b539158829 . _:b539158830 . . _:b539158831 . _:b539158907 "2"^^ . _:b539158824 . _:b539158898 . _:b539158825 . _:b539158826 . _:b539158827 . _:b539158906 "2"^^ . _:b539158820 . _:b27063090 "In the case of IL-1, specific antibody neutralization has been used to distinguish IL-1\u03B1 from IL-1\u03B2 (>>15<<, 16)." . _:b539158909 . _:b539158904 . _:b539158821 . _:b539158822 . _:b539158823 . _:b27063055 . _:b539158816 . _:b539158908 . _:b539158817 . _:b539158818 . _:b539158819 . _:b539158844 . _:b27063095 "Nevertheless, in vivo the IL-1\u03B2 precursor can be processed extracellularly by proteinase-3 and elastase into an active cytokine (>>9<<, 35). There is no caspase-1 cleavage site in the IL-1\u03B1 precursor; cleavage is by a membrane associated calpain results in a 17,000\u2009Da peptide. The N-terminus of the calpain cleavage product is assumed to be the same as that the IL-1\u03B1" . _:b539158911 . _:b539158845 . . _:b539158846 . _:b539158847 . _:b539158792 . _:b539158840 . _:b539158910 . _:b539158841 . _:b539158842 . _:b539158765 . _:b539158720 . _:b539158843 . _:b27063077 . _:b539158836 . _:b539158905 . _:b539158837 . . _:b539158838 . _:b539158839 . _:b27063063 . _:b539158913 "2"^^ . . _:b539158904 . _:b539158832 . _:b539158833 . _:b539158838 . _:b539158834 . _:b539158835 . _:b539158912 "2"^^ . _:b539158907 . _:b27063066 "In acute lung injury, the type II epithelial cells contain constitutive IL-1\u03B1 precursor and when released, mediates local inflammation, which is prevented by IL-1 receptor blockade (13, >>14<<). In a mouse model of DAMP activity from necrotic tumor cells injected intraperitoneally, there is neutrophilic inflammation but also elevation of hepatic enzyme alanine aminotransferase (ALT) (15). The inflammation was observed in" . . _:b539158906 . _:b539158906 . _:b27063057 . _:b539158701 . . _:b27063056 . _:b539158890 . _:b539158753 . _:b539158841 . _:b539158721 . _:b539158703 . . _:b27063058 . . _:b539158716 . _:b539158751 . _:b539158819 . _:b539158847 . _:b539158702 . . _:b539158913 . . _:b539158912 . _:b539158873 . _:b27063071 "Moreover, in vivo neutrophilic response to these unfractionated supernatants was blocked by an IL-1\u03B1 specific neutralizing antibody (>>16<<), suggesting that the IL-1\u03B1 precursor is active." . _:b539158742 . _:b27063109 . _:b539158757 . . _:b539158911 . _:b539158709 . . _:b27063101 "For example, others have reported that cellular contents release by high heat (>>15<<) or prolonged exposure to hypoxic conditions (4, 16) are due to IL-1\u03B1, not IL-1\u03B2 and not to TLR." . . _:b539158880 . _:b539158708 . . . _:b539158899 . _:b539158892 . . _:b539158711 . . . . _:b539158710 . . . _:b539158865 . _:b539158718 . _:b539158705 . _:b539158704 . _:b539158897 . . _:b539158707 . . _:b27063070 . . . _:b539158706 . _:b27063081 "different recombinant IL-1 family cytokines (precursor IL-1\u03B1, mature IL-1\u03B1, precursor IL-1\u03B2, mature IL-1\u03B2, precursor IL-33, and mature IL-33) and purified the recombinant proteins by two-steps of chromatography as described previously (>>6<<). Each recombinant cytokine was examined for purity by 10% SDS-PAGE and silver staining, and all six cytokines appeared as primarily a single band (Figure 1)." . _:b539158845 . . . _:b539158717 . _:b539158912 . _:b539158905 . . _:b539158913 . _:b27063072 "Since IL-1, either IL-1\u03B1 or IL-1\u03B2, promotes IL-17 production by T cells, alarmin-mediated immune responses may be an important endogenous activator of pathogenic T cells in autoimmune diseases (>>17<<, 18)." . _:b27063058 "Although during apoptosis, the intracellular IL-1\u03B1 precursor binds tightly to chromatin (4), during necrosis, the precursor dissociates from the chromatin is released from the cell in the precursor form (>>4<<). The IL-33 precursor also acts as a transcription factor and is chromatin bound (5); IL-33 is also released from dying cells where extracellular processing takes place by serine proteases (6, 7). The precursor forms for IL-1\u03B2 and IL-18" . _:b539158716 . _:b539158866 . _:b539158719 . _:b539158718 . _:b539158867 . _:b539158785 . _:b539158902 . _:b539158732 . _:b539158910 . _:b539158713 . _:b539158712 . . . . _:b539158715 . _:b27063109 . _:b539158740 . . _:b539158744 . _:b539158714 . _:b539158725 . _:b27063057 "Although during apoptosis, the intracellular IL-1\u03B1 precursor binds tightly to chromatin (>>4<<), during necrosis, the precursor dissociates from the chromatin is released from the cell in the precursor form (4)." . _:b539158724 . _:b539158786 . _:b539158857 . . _:b539158727 . _:b539158796 . . _:b539158708 . _:b539158726 . _:b539158721 . . _:b539158907 . _:b539158720 . _:b539158723 . . _:b539158711 . _:b539158741 . _:b539158722 . _:b539158733 . _:b539158877 . _:b539158732 . _:b539158784 . _:b27063111 . _:b27063105 "Here the release of apoptotic bodies released dying human endothelial cells contain the precursor as well as a processed form of IL-1\u03B1 (>>38<<). These apoptotic bodies are highly inflammatory in vivo and are due to IL-1\u03B1 and not IL-1\u03B2 (38). Another role for inflammation due to the IL-1\u03B1 precursor is in dying tumors. As tumors outgrow their vascular supply, necrosis takes place" . _:b539158735 . _:b539158879 . _:b539158734 . _:b539158781 . _:b539158729 . _:b539158714 . _:b539158728 . _:b539158733 . _:b27063085 . _:b539158731 . . . . _:b539158730 . _:b539158741 . _:b539158886 . _:b539158740 . _:b27063087 . _:b539158737 . . _:b27063069 "Furthermore, antibodies to IL-1\u03B1 rather than to IL-1\u03B2 blocked the response and the inflammation was still observed in mice deficient in TLR (>>15<<). In those studies, the molecular nature of the IL-1\u03B1, i.e., whether the precursor or the mature form, was not determined. However, in another study (4) keratinocytes exposed to prolonged hypoxia in vitro released cellular contents" . _:b539158734 . . . _:b539158743 . _:b27063093 "The study identified several sequences along one face of the molecule which are distinct from regions thought to bind to the IL-1 receptor (>>33<<). In the present study, we compared the biological activity of recombinant IL-1\u03B1, IL-1\u03B2, and IL-33 precursors to the activity as mature proteins using a biologically relevant assay of cytokine production from human lung epithelial A549" . . _:b27063106 . . _:b539158742 . _:b539158723 . _:b27063083 . _:b27063064 "Pathogen-associated molecular patterns (PAMPs) initiate inflammation by an infectious pathogen, most commonly via Toll-Like Receptors (TLR) (>>12<<)." . _:b539158737 . . _:b539158706 . _:b539158736 . . _:b27063105 . _:b27063110 "form of IL-33 is not active on mast cells compared to the processed forms; it is likely that IL-33 has limited activities due to its receptor expression pattern in different immune cell linages including non-immune cell linage (>>40<<). Although precursor IL-33 possesses biological activity, unlike precursor IL-1\u03B1, the impact of effects could be less because only particular immune cells linages respond to IL-33." . _:b539158739 . _:b27063102 . _:b539158738 . _:b539158846 . . _:b27063084 . . _:b539158749 . _:b539158828 . _:b27063087 . . _:b27063088 "discussion" . . _:b539158748 . _:b27063074 "materials and methods" . _:b539158751 . _:b27063088 _:b27063092 . _:b539158756 . _:b27063088 _:b27063093 . _:b27063075 "The recombinant proteins were expressed in E. coli with six N-terminal histidine tags and purified over a mini-Talon chromatography, ion-exchange for IL-1\u03B2 or HPLC for IL-1\u03B1 and IL-33 as described (>>6<<). Purity was assessed by silver staining of PAGE (Figure 1). The endotoxin content was less that 1\u2009EU/mg." . _:b539158750 . . _:b27063088 _:b27063094 . _:b27063088 _:b27063095 . _:b27063081 . _:b539158826 . _:b27063078 "The human mast cell responses to IL-33 were measured in the HMC-1 cell line as described previously (>>6<<, 19). After 24\u2009h of incubation with mature or precursor IL-33, IL-8 was measured in the supernatants (R&D Systems)." . _:b27063088 _:b27063089 . _:b539158745 . _:b27063088 _:b27063090 . . _:b27063088 _:b27063091 . _:b27063088 _:b27063100 . _:b27063088 _:b27063101 . _:b539158744 . _:b27063088 _:b27063102 . _:b539158702 . _:b27063088 _:b27063103 . _:b539158705 . _:b27063088 _:b27063096 . . _:b539158747 . . _:b27063088 _:b27063097 . _:b27063088 _:b27063098 . _:b27063088 _:b27063099 . . _:b539158746 . . _:b539158757 . _:b539158834 . _:b539158840 . _:b539158756 . . _:b539158862 . _:b539158759 . _:b539158856 . _:b27063061 . _:b539158758 . _:b27063056 . _:b539158753 . . _:b539158777 . . _:b539158790 . . _:b539158752 . . _:b27063087 . _:b539158732 . . _:b27063075 . _:b539158872 . _:b539158733 . _:b539158734 . _:b539158735 . _:b539158912 . _:b539158755 . _:b539158728 . _:b539158729 . _:b539158730 . _:b539158731 . _:b539158754 . _:b539158724 . _:b539158725 . _:b539158726 . _:b539158820 . . _:b539158765 . _:b539158727 . _:b539158720 . "PMC0" . _:b27063078 . _:b539158895 . _:b539158721 . _:b27063074 _:b27063076 . _:b539158722 . _:b27063074 _:b27063077 . _:b539158764 . _:b539158723 . _:b27063074 _:b27063078 . _:b539158748 . _:b27063074 _:b27063079 . _:b539158749 . _:b539158750 . _:b539158760 . _:b539158751 . _:b539158767 . _:b539158744 . _:b27063074 _:b27063075 . _:b539158745 . _:b539158746 . _:b539158747 . _:b27063109 . _:b539158766 . _:b539158740 . _:b539158741 . _:b539158742 . _:b539158743 . _:b539158761 . . _:b539158736 . _:b539158737 . _:b539158821 . . _:b539158738 . _:b539158739 . _:b539158760 . _:b539158764 . _:b539158765 . _:b539158766 . . _:b539158763 . _:b539158767 . _:b539158760 . _:b539158761 . . _:b539158762 . _:b27063088 _:b27063108 . _:b27063088 _:b27063109 . _:b539158762 . _:b539158763 . _:b27063088 _:b27063110 . _:b539158783 . . _:b539158756 . _:b27063088 _:b27063111 . _:b539158757 . _:b539158702 "8"^^ . _:b539158758 . _:b27063088 _:b27063104 . . _:b27063088 _:b27063105 . _:b539158773 . _:b539158759 . _:b27063088 _:b27063106 . _:b539158752 . _:b27063088 _:b27063107 . _:b539158900 . _:b539158753 . _:b539158703 "8"^^ . _:b539158754 . _:b27063080 _:b27063084 . _:b27063080 _:b27063085 . _:b27063089 . _:b539158772 . _:b539158755 . _:b27063080 _:b27063086 . _:b539158780 . _:b27063080 _:b27063087 . _:b27063099 "In the case of the precursor forms of IL-1\u03B1 and IL-33, N-terminal pro-sequence has another function, binding to nucleic DNA and therefore participating in transcription (>>3<<, 5)." . _:b539158781 . _:b27063088 _:b27063112 . _:b539158782 . _:b27063088 _:b27063113 . _:b539158844 . _:b539158775 . _:b539158783 . _:b27063080 _:b27063081 . _:b27063080 _:b27063082 . _:b539158835 . _:b539158776 . . _:b27063080 _:b27063083 . _:b539158777 . _:b539158701 "8"^^ . _:b539158730 . _:b539158778 . _:b539158779 . _:b539158774 . _:b539158772 . _:b539158773 . _:b539158817 . _:b539158774 . . _:b539158769 . _:b539158775 . _:b539158768 . _:b539158769 . . _:b539158770 . _:b539158771 . _:b539158768 . _:b539158767 . . . _:b539158771 . . . _:b539158752 . _:b539158770 . . _:b539158781 . . _:b27063063 "Because DAMP\u2019s rapidly initiate inflammation cell in the absence of proper secretion mechanisms, the term \u201Calarmin\u201D is often used for categorizing such endogenous molecule as proposed by Oppenheim (>>11<<). The released intracellular proteins have immediate effects on cells expressing receptors involved in inflammation, host defense, and tissue repair. Pathogen-associated molecular patterns (PAMPs) initiate inflammation by an infectious" . _:b539158780 . _:b539158708 "6"^^ . _:b539158807 . _:b539158711 "5"^^ . _:b539158783 . . _:b539158709 "6"^^ . _:b539158710 "5"^^ . _:b539158782 . _:b539158706 "6"^^ . _:b27063098 . . _:b539158777 . _:b539158707 "6"^^ . _:b539158874 . . _:b539158776 . _:b539158704 "7"^^ . _:b539158701 . _:b539158702 . _:b539158703 . _:b27063099 . _:b539158779 . _:b539158705 "7"^^ . . _:b539158778 . _:b539158848 . _:b27063059 "The IL-33 precursor also acts as a transcription factor and is chromatin bound (>>5<<); IL-33 is also released from dying cells where extracellular processing takes place by serine proteases (6, 7)." . _:b27063072 . _:b539158717 "4"^^ . . . _:b539158833 . _:b539158716 "4"^^ . _:b27063097 "The N-terminus of the calpain cleavage product is assumed to be the same as that the IL-1\u03B1 purified from the supernatants of a mouse cell line (>>36<<). However, it remains unclear how calpain is activated. For IL-33, extracellular processing by serine proteases generate several N-termini with varying degrees of activity (7). In the case of the precursor forms of IL-1\u03B1 and IL-33," . _:b539158739 . _:b539158755 . . _:b539158716 . _:b539158717 . _:b27063092 . _:b539158903 . _:b539158719 "4"^^ . _:b539158718 . . _:b539158719 . . _:b539158712 . _:b539158713 . _:b539158802 . _:b539158718 "4"^^ . _:b539158714 . _:b539158715 . _:b539158708 . _:b539158815 . _:b539158709 . _:b539158710 . _:b27063113 "In other studies, IL-33 exhibits a weak activity as a precursor compared to mature IL-33 (6, 19, >>40<<)." . _:b539158713 "5"^^ . _:b27063094 . _:b539158780 . _:b539158711 . . _:b539158704 . . _:b539158759 . _:b539158705 . _:b539158706 . _:b539158712 "5"^^ . _:b539158707 . _:b539158715 "4"^^ . _:b539158794 . _:b27063060 "The IL-33 precursor also acts as a transcription factor and is chromatin bound (5); IL-33 is also released from dying cells where extracellular processing takes place by serine proteases (>>6<<, 7). The precursor forms for IL-1\u03B2 and IL-18 are inactive; processing to an active cytokine can take place intracellularly by caspase-1 but extracellularly by neutrophil-derived proteinase-3 (8\u201310)." . _:b27063054 _:b27063068 . _:b539158714 "5"^^ . _:b27063054 _:b27063069 . _:b27063054 _:b27063070 . _:b27063054 _:b27063071 . _:b27063085 "IL-1 receptor antagonist (anakinra) is used to treat a broad spectrum of acute and chronic inflammatory diseases, as reviewed in Ref. (>>23<<) and since IL-1Ra blocks the IL-1R1, it remains unknown how many diseases that are controlled by anakinra are due to prevention of IL-1\u03B1 or IL-1\u03B2 activity; furthermore, how many diseases are due to the activity of the IL-1\u03B1 precursor?" . _:b539158725 "4"^^ . _:b27063054 _:b27063064 . _:b27063054 _:b27063065 . _:b27063054 _:b27063066 . _:b27063054 _:b27063067 . _:b539158724 "4"^^ . _:b27063054 _:b27063060 . _:b27063054 _:b27063061 . _:b27063054 _:b27063062 . . _:b27063054 _:b27063063 . _:b539158707 . _:b539158727 "4"^^ . _:b27063054 _:b27063056 . _:b27063054 _:b27063057 . . _:b27063054 _:b27063058 . _:b27063054 _:b27063059 . _:b539158726 "4"^^ . _:b27063054 _:b27063055 . _:b27063097 . _:b539158721 "4"^^ . _:b27063061 "The IL-33 precursor also acts as a transcription factor and is chromatin bound (5); IL-33 is also released from dying cells where extracellular processing takes place by serine proteases (6, >>7<<). The precursor forms for IL-1\u03B2 and IL-18 are inactive; processing to an active cytokine can take place intracellularly by caspase-1 but extracellularly by neutrophil-derived proteinase-3 (8\u201310)." . _:b539158720 "4"^^ . _:b539158831 . . _:b539158812 . _:b539158888 . _:b539158723 "4"^^ . _:b27063076 . . _:b539158738 . _:b539158843 . _:b27063098 "For IL-33, extracellular processing by serine proteases generate several N-termini with varying degrees of activity (>>7<<). In the case of the precursor forms of IL-1\u03B1 and IL-33, N-terminal pro-sequence has another function, binding to nucleic DNA and therefore participating in transcription (3, 5)." . _:b539158722 "4"^^ . _:b27063079 . _:b539158788 . _:b539158733 "3"^^ . _:b539158732 "3"^^ . _:b539158735 "3"^^ . _:b539158736 . _:b27063111 "In other studies, IL-33 exhibits a weak activity as a precursor compared to mature IL-33 (>>6<<, 19, 40)." . . _:b539158734 "3"^^ . . _:b539158853 . _:b539158729 "4"^^ . _:b539158878 . . . _:b539158728 "4"^^ . _:b27063087 . _:b539158858 . . _:b539158747 . _:b539158731 "3"^^ . _:b27063054 _:b27063072 . _:b27063054 _:b27063073 . _:b27063060 . _:b539158730 "4"^^ . _:b539158803 . _:b539158741 "3"^^ . _:b27063103 "For example, others have reported that cellular contents release by high heat (15) or prolonged exposure to hypoxic conditions (4, >>16<<) are due to IL-1\u03B1, not IL-1\u03B2 and not to TLR." . _:b539158740 "3"^^ . _:b27063104 . _:b539158743 "3"^^ . _:b27063102 "For example, others have reported that cellular contents release by high heat (15) or prolonged exposure to hypoxic conditions (>>4<<, 16) are due to IL-1\u03B1, not IL-1\u03B2 and not to TLR." . _:b27063107 . _:b539158742 "3"^^ . _:b539158876 . _:b539158737 "3"^^ . _:b539158728 . . _:b539158823 . _:b539158736 "3"^^ . . . _:b27063108 . _:b539158739 "3"^^ . . . _:b539158825 . _:b27063094 "The IL-1\u03B2 precursor was consistently without activity on A549 cells and PBMC whereas the mature form with the N-terminus at the caspase-1 cleavage site (>>34<<) was active. Nevertheless, in vivo the IL-1\u03B2 precursor can be processed extracellularly by proteinase-3 and elastase into an active cytokine (9, 35)." . _:b539158738 "3"^^ . _:b539158749 "3"^^ . _:b539158908 . _:b27063068 "The inflammation was observed in wild-type (WT) mice but not in mice deficient in the IL-1 receptor type 1 (IL-1R1) (>>15<<). Furthermore, antibodies to IL-1\u03B1 rather than to IL-1\u03B2 blocked the response and the inflammation was still observed in mice deficient in TLR (15). In those studies, the molecular nature of the IL-1\u03B1, i.e., whether the precursor or the" . . _:b539158748 "3"^^ . _:b539158829 . _:b539158751 "3"^^ . _:b27063087 . _:b539158750 "3"^^ . _:b539158703 . _:b539158745 "3"^^ . . _:b27063067 "In a mouse model of DAMP activity from necrotic tumor cells injected intraperitoneally, there is neutrophilic inflammation but also elevation of hepatic enzyme alanine aminotransferase (ALT) (>>15<<). The inflammation was observed in wild-type (WT) mice but not in mice deficient in the IL-1 receptor type 1 (IL-1R1) (15). Furthermore, antibodies to IL-1\u03B1 rather than to IL-1\u03B2 blocked the response and the inflammation was still observed" . _:b539158744 "3"^^ . . _:b539158747 "3"^^ . _:b27063084 "Since IL-1 induces TNF\u03B1 in vivo and in vitro (>>24<<), we next examined the induction of TNF\u03B1 by precursor and mature forms of IL-1\u03B1 and IL-1\u03B2 on PBMC." . _:b539158746 "3"^^ . _:b539158757 "3"^^ . _:b539158861 . . _:b539158756 "3"^^ . _:b539158704 . _:b539158875 . _:b27063062 . _:b539158759 "3"^^ . _:b27063092 . _:b539158758 "3"^^ . . _:b539158753 "3"^^ . _:b539158868 . _:b539158752 "3"^^ . _:b27063083 "whether the human monoclonal antibody to IL-1\u03B1 neutralized the precursor form of IL-1\u03B1. This antibody is presently be studied in several clinical trials, including type-2 diabetes, psoriasis, and cancer cachexia, as reviewed in Ref. (>>23<<). Precursor or mature IL-1\u03B1 at 0.5\u2009nM was preincubated for 30\u2009min with increasing molar excesses of anti-IL-1\u03B1 (XBiotech, Austin, TX, USA)." . _:b27063109 . _:b539158755 "3"^^ . _:b27063082 . _:b539158754 "3"^^ . . _:b539158765 "3"^^ . . . _:b539158883 . . . _:b539158764 "3"^^ . . . _:b27063086 . _:b539158767 "3"^^ . . _:b539158860 . _:b539158804 . _:b539158766 "3"^^ . _:b27063109 "Since is released from tumor cells and necrotic cells in the context of intracellular cytokines, IL-33 can be seen as a novel of alarmin (>>26<<\u201331). However, our data (Figure 6) show that the precursor form of IL-33 is not active on mast cells compared to the processed forms; it is likely that IL-33 has limited activities due to its receptor expression pattern in different immune" . . _:b539158761 "3"^^ . _:b539158830 . . _:b539158760 "3"^^ . _:b539158859 . _:b539158763 "3"^^ . _:b539158727 . _:b539158762 "3"^^ . . _:b539158824 . . _:b27063089 "cell surface receptors; these include high mobility group box-1 (HMGB-1), members of the IL-1 family, cytosolic calcium-binding proteins of the S100 family, heat-shock proteins 70/90, S100 proteins, peroxiredoxin-1, and nucleosomes (>>32<<). For example, HMGB-1 binds to the cell surface scavenger receptor (RAGE) and IL-1\u03B1 binds to the IL-1R1." . _:b539158773 "2"^^ . . _:b539158772 "2"^^ . . _:b539158775 "2"^^ . _:b539158774 . _:b539158774 "2"^^ . _:b539158762 . .