Emiko Sano

Identification of interleukin-8 converting enzyme as cathepsin L

IL-8 is produced by various cells, and the NH(2)-terminal amino acid sequence of IL-8 displays heterogeneity among cell types. The mature form of IL-8 has 72 amino acids (72IL-8), while a precursor form (77IL-8) of IL-8 has five additional amino acids to the 72IL-8 NH(2)-terminal. However, it has been unclear how IL-8 is processed to yield the mature form. In this study, converting enzyme was purified as a single 31-kDa band on silver-stained polyacrylamide gel from 160 l of cultured fibroblast supernatant by sequential chromatography. NH(2)-terminal amino acid sequence analysis revealed a sequence, EAPRSVDWRE, which was identified as a partial sequence of cathepsin L. Polyclonal antibodies raised against cathepsin L recognized the purified converting enzyme on Western blot. Moreover, human hepatic cathepsin L cleaved 77IL-8 between Arg(5) and Ser(6), which is the same cleavage site as the putative converting enzyme, resulting in 72IL-8 formation. These data indicate that the converting enzyme of the partially purified fraction of the human fibroblast culture supernatant was cathepsin L. Furthermore, 72IL-8 was sevenfold more potent than 77IL-8 in a neutrophil chemotaxis assay. These results show that cathepsin L is secreted from human fibroblasts in response to external stimuli and plays an important role in IL-8 processing in inflammatory sites.

Structural Characterization of Recombinant Human Interferon-Gammas Derived from Two Different Mammalian Cells

Recombinant human interferon‐gammas (rHuIFN‐γs) were obtained from two different mammalian cells (mouse C127 cells and Chinese hamster ovary, CHO, cells) cultured in a microcarrier culture system. Both rHuIFN‐γs were purified using sequential chromatographies for their comparison of structural properties. The peptide maps of HuIFN‐γs digested with V8 protease and Western blot analysis demonstrated that C127 cells yielded mainly about 25kDa component and CHO cells produced about 25kDa and about 20kDa components. By the identification of glycosylated peptides, it was suggested that 20kDa and 25kDa components are glycosylated at one and at two sites, respectively. C‐terminal amino acid sequence analysis indicated that both rHuIFN‐γs consisted of at least six different species lacking 2 to 16 amino acid residues from C‐terminus, so that C‐termini of both rHuIFN‐γs were slightly different from each other.

A possible role of autogenous IFN-β for cytokine productions in human fibroblasts

It has been already known that human diploid fibroblasts are able to produce not only high levels of IFN‐β but also various kinds of cytokines by poly rI: poly rC, and some inflammatory cytokines are induced by IFN‐β gene activation. We also obtained similar results. However, in our system, cytokine productions were extremely enhanced by treating the cells with a low dose of type 1 IFN and the priming effects on cytokine productions were blocked by cycloheximide similar to those on IFN‐β productions. Most of cytokines were produced later than IFN‐β and synthesis patterns of their mRNA showed the same phenomena. We made clear that cytokine productions by poly rI: poly rC are mediated by secreted IFN‐β at a protein level using a monoclonal antibody against human IFN‐β. Further, it was shown that intra‐cellular IFN‐β which is not secreted might also participate in cytokine productions. Meanwhile, IL‐1β induced various kinds of cytokines in human fibroblasts and production time courses of these cytokines were similar to those of poly rI: poly rC induced cytokines. Although secreted IFN‐β was not detected in IL‐1β stimulated culture, expression of IFN‐β mRNA was augmented. These results showed that priming effects of type 1 IFN on cytokine productions by poly rI: poly rC might not be the direct action, but successive IFN‐β production might be essential in the production processes of other cytokines. Further, it was suggested that inducible IFN‐β might also take part in IL‐1β‐induced cytokine productions. J. Cell. Biochem. 100: 1459–1476, 2007.

Possible identity of IL-8 converting enzyme in human fibroblasts as a cysteine protease

A converting activity was characterized in human diploid fibroblasts, which secrete 72IL-8 and 77IL-8 in treatment with IFN-beta and poly I: poly C. 77IL-8 was significantly converted to 72IL-8 by a partially purified fraction of the culture supernatant of human diploid fibroblasts. The converting activity, which was temperature-dependent and optimal at pH 6, was completely inhibited by cysteine protease inhibitors, antipain dihydrochloride and E-64, but not by other types of protease inhibitors. These data clearly show that human diploid fibroblasts are capable of processing IL-8 to produce a mature IL-8 and that the putative converting enzyme appears to be a cysteine protease.

Constitutive Long-Term Production of Recombinant Human Interferon-Gamma by Transformed Mouse C127 Cells Cultured in Serum Free Medium

Transformed mouse C127 cells in which human interferon-gamma cDNA was inserted were grown well in a microcarrier culture system. The cells were maintained for more than two months even in a serum free condition without cell proliferation and continued to produce a high level of recombinant human interferon-gamma (rHuIFN-r). The maintenance of the cells grown on the microcarriers was fairly prolonged by adding 0, 1% bovine serum albumin to the serum free medium. The flow-cytometrical analysis for cell cycle showed more than 85% of these maintained cells remained in G0/G1 phase, differing from the case of the growing cells. However, we confirmed the copy-numbers of rHuIFN-r cDNA were safely retained even in the maintained cells for a long period. The rHuIFN-r preparation obtained in the serum free condition was also glycosylated protein similar to natural HuIFN-r derived from human periferal blood lymphocytes.