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Leukotactin-1 (Lkn-1), a human CC chemokine that binds to both CC chemokine receptor (CCR)1 and CCR3, is distinct from other human CC chemokines in that it has long amino acid residues preceding the first cysteine at the NH(2)-terminus. Serial deletion studies showed that at least three amino acid residues, alanine-alanine-aspartic acid (A-A-D), preceding the first cysteine at the NH(2)-terminus are essential for the biological activity of Lkn-1. Point mutation and deletion studies for the three amino acids were performed in the present study. Substitutions of the first alanine residue with other amino acids did not cause significant loss of biological activities. Deletion of the third amino acid, aspartic acid, resulted in more than 100-fold loss of the activity. Deletion of two amino acids, alanine-alanine (A-A) or alanine-aspartic acid (A-D), resulted in almost complete loss of the activity. Loss of agonistic activity by deletion of two amino acids was due to impaired binding to CCR1. These results identify that alanine-aspartic acid residues preceding the first cysteine at the NH(2)-terminus are essential for the binding and biological activity of Lkn-1.

The herb, Chrysanthemum zawadskii var, latilobum commomly known as Gu-Jul-Cho in Korea, used in traditional medicine to treat pneumonia, bronchitis, cough, common cold, pharyngitis, bladder-related disorders, gastroenteric disorders, and hypertension. Linarin is the main active compound and the biological mechanisms of its activity are unclear. It is believed that effects of this herb may be exerted through the pluripotent effectors of linarin due to its ability to treat a variety of afflictions. In this study, the effects of linarin on the mouse macrophages cell line, RAW 264.7, were investigated. It was found that linarin could activate macrophages by producing cytokines. Monocytes and tissue macrophages produce at least two groups of protein mediators of inflammation, interleukin 1 (IL-1) and the tumor necrosis factor (TNF). Recent studies have shown that TNF and IL-1 modulate the inflammatory function of endothelial cells, leukocytes, and fibroblasts. TNF-alpha production by macrophages treated with linarin occured in a dose dependent manner. However, IL-1 production was largely unaffected by this natural product. This study demonstrated the ability of linarin to activate macrophages both directly and indirectly. Linarin also affect both cytokine production and nitric oxide inhibition, in addition to the expression of some surface molecules. Nitric oxide (NO), derived from L-argin-ine, is produced by two forms(constitutive and inducible) of nitric oxide synthase (NOS). The NO produced in large amounts by inducible NOS is known to be responsible for the vasodilation and hypotension observed in septic shock. Linarin was found to inhibit NO production in the LPS-activated RAW 264.7 cells. Linarin may be a useful candidate as a new drug for treating endotoxemia and the inflammation accompanied by NO overproduction. The linarin-treated total lymphocytes exhibited cytotoxicity in a dose dependent manner between 20 microg/ml and 40 microg/ml. These results suggest that linarin may function through macrophage activation.