Both interleukin‐8 receptors independently mediate chemotaxis

Abstract

Neutrophil leukocytes, the target cells for interleukin-8 and related CXC chemokines, bear high numbers of two types of IL-8 receptors (IL-8Rl and IL-8R2). By cDNA transfection Jurkat cell lines were generated that stably express either IL-8Rl or IL-8R2 (J-IL8Rl and J-IL8R2). J-IL8Rl expressed 4,000 + 1,000 copies of IL-8R1, and bound IL-8 with high affinity (Kd l-4 nM) and GROcl and NAP-2 with low affinity (Kd 200-500 nM). J-IL8R2 expressed 17,000 f 3,000 copies of IL-8R2, and bound all three chemokines with high affinity. Both transfectants showed a similar degree of chemotactic migration after stimulation with IL-8, GROa and NAP-2. All three chemokines were equally potent as attractants of J-IL8R2, whereas IL-8 was 300 to l,OOO-fold more potent than GROcl or NAP-2 as attractant of J-IL8Rl. The potencies, therefore, agree with the affinities of the ligands to IL-8Rl and IL-8R2. Our results demonstrate that both IL-8 receptors function independently, and mediate chemotaxis in response to IL-8 and other CXC chemokines.

A comparison of post‐receptor signal transduction events in Jurkat cells transfected with either IL‐8R1 or IL‐8R2 Chemokine mediated activation of p42/p44 MAP‐kinase (ERK‐2)

“…Both cell lines were found to bind IL-8 with similar high affinity (Table 1), comparable with IL-8 binding to human neutrophils [7,9]. Although, Loetscher et al [20] previously demonstrated the migratory characteristics of Jurkat cells transfected with IL-8 receptors, other chemokine-induced neutrophil functions were not addressed. Here we were unable to detect IL-8 stimulation of superoxide production in our transfectants and Western blot analysis of Jurkat cell lysates confirmed the absence of p47-phox and p67-phox, cytosolic factors of the superoxide-generating NADPH oxidase, (data not shown).…”

“…A 1,112 bp cDNA fragment encoding for IL-8R1 was generated as outlined by Leotscher et al [20] and sub-cloned into pcDNA-Y Briefly, 20/tg oflinearised pcDNA-3/IL-8R 1 was transfected into Jurkat cells (5-8 x 106 cells/800/.tl PBS) by electroporation. Two weeks later G418-resistant (0.8 mg/ml) Jurkat clones were selected by limited dilution and characterized for expression of IL-8R 1 by ~25I-IL-8 binding.…”

“…Of four independent clones, the clone with the highest binding capacity was selected and termed J-IL8R1. Jurkat cells transfected with cDNA encoding for human IL-8R2 were generated as previously described by Leotscher et al [20] and is refered to here as J-IL8R2.…”

“…Calcium mobilisation and in vitro chemotaxis assays were performed as previously described by Loetscher et al [20].…”

“…This suggests that both receptors not only possess different ligandbinding properties but may also initiate different post-receptor signal transduction events. Leotscher et al [20] …”