Isolation and amino acid sequence of a chemotactic protein, LECT/interleukin 8, from a human myeloid leukemia cell line, ML-1.
We looked for chemotaxin/interleukin 8 (CT/IL-8) activity in the culture fluids of 97 human leukemia cell lines and found it in two of the T cell lines, six of the myeloid cell lines, and one of the normal B-cell lines. It was particularly strong in the culture fluids of two cell lines. These cell lines secreted a chemotactic protein into the culture fluids under certain conditions of stimulation with phorbol-12-myristate 13-acetate (PMA), lipopolysaccharide, or hemagglutinin-P. A myeloid leukemia cell line, ML-1, secreted an inducible chemotaxin when stimulated with PMA (1 ng/ml) for 24 h. We purified the chemotaxin from ML-1 cell culture fluid using an improved procedure: concentration with DEAE-Sepharose CL-6B and CM-Sepharose CL-6B, CM-Sepharose column chromatography, and reverse-phase 5TMS-300 column on HPLC with the retention time coinciding with that of LUCT/IL-8 [Suzuki et al., 1989, J. Exp. Med. 169, 1895]. The yield was 200 micrograms protein from 6 liters of the culture fluid. The N terminus of CT/IL-8 was AVLPR-SAKELRXQXIKTYSK- - -, the same as that of LUCT/IL-8, which is constitutively secreted from lung giant cell carcinoma LU65C cells. The optimal concentration in the chemotactic activity of CT/IL-8, equivalent to that of bacterial chemotactic peptide fMet-Leu-Phe (10 nM), was found to be 5 nM. The results show that this chemotaxin is identical to LUCT/IL-8.
Suzuki
K
K
Department of Bioactive Molecules, National Institute of Health, Tokyo, Japan.
Yamakawa
Y
Y
Matsuo
Y
Y
Kamiya
T
T
Minowada
J
J
Mizuno
S
S
D000595:Amino Acid Sequence; D001402:B-Lymphocytes / Q000276:immunology; D002630:Chemotactic Factors / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D002634:Chemotaxis, Leukocyte / Q000276:immunology; D002851:Chromatography, High Pressure Liquid; D006801:Humans; D016209:Interleukin-8 / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D007951:Leukemia, Myeloid / Q000276:immunology*; D008969:Molecular Sequence Data; D009504:Neutrophils / Q000276:immunology; D017386:Sequence Homology, Amino Acid; D013601:T-Lymphocytes / Q000276:immunology; D014407:Tumor Cells, Cultured
D002630:Chemotactic Factors; D016209:Interleukin-8