Stimulation of Interleukin-8 Production in Human THP-1 Macrophages by Apolipoprotein(a)

Abstract

In the vessel wall, macrophages are among the cells that upon activation contribute to the atherosclerotic process. Low density lipoproteins (LDL) can mediate this activation but only after enzymatic or oxidative modification. Lipoprotein(a) (Lp(a)) is an LDL variant that has been shown to have an atherogenic potential by no clearly established mechanisms. In the present study we examined whether native Lp(a) can activate macrophages and, if so, identify the structural elements involved in this action. For this purpose, we utilized human THP-1 macrophages, prepared by treating THP-1 monocytes with phorbol ester, and we exposed them to Lp(a) and its two derivatives, apo(a)-free LDL (Lp(a؊)) and free apo(a). We also studied apo(a) fragments, F1 (N terminus) and F2 (C terminus) and subfragments thereof, obtained by leukocyte elastase digestion. By Northern blot analyses, Lp(a), but not Lp(a؊), caused up to a 12-fold increase in interleukin 8 (IL-8) mRNA as compared with untreated cells. Free apo(a) also induced the production of IL-8 mRNA; however, the effect was 3-4-fold higher than that of Lp(a). The increase in mRNA was associated with the accumulation of IL-8 protein in the culture medium. F1 had only a minimal effect, whereas F2 was 1.5-2-fold more potent than apo(a), an activity mostly contained in the Kringle V-protease region. A monoclonal antibody specific for Kringle V inhibited the apo(a)-mediated effect on IL-8. We conclude that Lp(a) via elements contained in the C-terminal domain of apo(a) causes in THP-1 macrophages an increased production of IL-8, a chemokine with pro-inflammatory properties, an event that may be relevant to the process of atherosclerosis.

Lysine-Phosphatidylcholine Adducts in Kringle V Impart Unique Immunological and Potential Pro-inflammatory Properties to Human Apolipoprotein(a)

“…Studies on THP-1 Cells in Culture-The conditions were those previously described (5). Briefly, the human monocyte leukemia cell line, THP-1, obtained from American Type Culture Collection (Manassas, VA) was maintained in RPMI 1640 medium supplemented with10% fetal bovine serum, penicillin (50 units/ml), streptomycin, and gentamicin (50 g/ml) and 50 M ␤ME at 37°C, 5% CO 2 .…”

“…1. The digestion of Lp(a) and the isolation of the fragments was as described previously (5). The purity of the fragments was assessed by SDS-PAGE on Coomassie-stained gels and by Western blot analysis using polyclonal monospecific rabbit anti-apo(a) and anti-apoB100 antibodies as well as monoclonal anti-KV antibodies.…”

“…For instance, the microdomain comprising KIV-5 through KV-8 has been reported to be involved in the first step of non-covalent interaction between apo(a) and apoB100, and KIV-9 via its unpaired cysteine is responsible for the formation of the disulfide bond that stabilizes the interaction between apo(a) and apoB100 (1,2). Moreover, KIV-10 contains the high affinity lysine binding site that plays a dominant role in the binding of apo(a) to fibrin(ogen) and components of the vascular extracellular matrix (3). For KV, the reported roles have been an attenuating effect on LDL oxidation (4) and stimulation of IL-8 production in human macrophages (5).…”

“…Moreover, KIV-10 contains the high affinity lysine binding site that plays a dominant role in the binding of apo(a) to fibrin(ogen) and components of the vascular extracellular matrix (3). For KV, the reported roles have been an attenuating effect on LDL oxidation (4) and stimulation of IL-8 production in human macrophages (5). Of interest, human KV has six lysine residues in contrast with the other kringles that contain no lysine except for KIV-4 and KIV-9 each having one lysine (6).…”

Interleukin-1β stimulates IL-8 expression through MAP kinase and ROS signaling in human gastric carcinoma cells

“…In addition to its potent chemotactic activity, it can induce proliferation and migration of keratinocytes, endothelial cells, and melanoma cells (Rennekampff et al, 2000;Singh and Varney, 2000;Heidemann et al, 2003). IL-8 is expressed in many different cell types, including monocytes and macrophages, endothelial cells, keratinocytes, mesangial cells, and several human tumor cell lines (Schwarz et al, 1997;Klezovitch et al, 2001;Maeda et al, 2001). Although constitutive production of IL-8 appears to be infrequent in most cell types examined, its expression can be induced in many cell types by specific stimuli such as granulocyte colony-stimulating factor, lipopolysaccharide (LPS), phorbol 12-myristate 13-acetate, viruses, and doublestranded RNA (Kasahara et al, 1991).…”