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The N- and C-terminal peptides of hIL8/CXCL8 are ligands for hCXCR1 and hCXCR2 ¹

Department of Cell Biology and Neuroscience, University of California, Riverside, California, USA

³Correspondence: Department of Cell Biology and Neuroscience, Spieth Hall, 900 University Ave., University of California, Riverside, CA 92521, USA. E-mail: mmgreen{at}ucrac1.ucr.edu

SPECIFIC AIM

It is known that peptide sequences corresponding to N- and C-terminal regions of certain chemokines belonging to the CXC family can stimulate specific functions in vivo and in vitro. The aim of this study was to determine whether these terminal peptides alone can activate specific chemokines receptors. We have used the two forms of human IL8 (hIL8/CXCL8) to link multifunctionality with peptides that represent various functional domains of these chemokines.

PRINCIPAL FINDINGS

Chemokine receptors are seven transmembrane proteins that couple to G_i-containing trimeric G-proteins. Binding of chemokines to their receptors triggers receptor activation and recruitment of G, followed by activation of PLC-ß and the influx of Ca²⁺ from smooth ER to the cytosol. To determine whether hCXCR1 is a receptor for the N- and C-terminal peptides of hIL8, we used Ca²⁺ mobilization and ERK1/2 phosphorylation/activation assays. We used NIH3T3 cells, taking advantage of the murine genetic background, which lacks the hCXCR1 gene. To obtain a homogeneous and controllable hCXCR1 expression, we used a Tet-on retroviral system for gene delivery. To demonstrate functionality we performed chemotaxis assays, including primary human microvascular endothelial cells that express both CXCR1 and CXCR2.

1. hIL8 N- and C-peptides stimulate intracellular Ca²⁺ release
To monitor intracellular Ca²⁺ levels upon ligand–receptor interaction, we cultured hCXCR1-expressing cells on glass coverslips and loaded the cells with the Ca²⁺ indicator Fluo-3AM for imaging. After choosing the field of cells to be examined, we established the baseline fluorescence (Fig. 1 A, C, E, G, left panels). Ligands were then individually delivered onto the cells by pressure ejection with N₂ from a glass micropipette positioned directly above the cells allowing measurement of the intracellular Ca²⁺ level increases for each ligand. All cells responded to hIL8-72 (Fig. 1A , right panel) and IL8-77 (Fig. 1C , right panel). Figure 1B, D shows that both hIL8s activate hCXCR1 in a typical chemokine receptor response with Ca²⁺ mobilization and that this response is dose dependent (negative controls: –Dox treatment).

View larger version (48K): [in this window] [in a new window]   Figure 1. hIL8 and its N- and C-peptides are ligands for hCXCR1. A) Representative images of Dox-treated cells in resting state and at peak Ca2+ response after exposure to hIL8-72. B) Average changes in Ca2+ concentration in the cytosol for cells imaged in panel A. dF/Fo averages of the selected cells were plotted against time. hIL8-72 induced rapid, strong and transient Ca2+ mobilization; cells in the absence of Dox treatment were insensitive to IL8. The straight solid line below the curves indicates the period of exposure to hIL8-72. C) Representative images of Dox-treated cells at rest and at the peak of intracellular Ca2+ increase after exposure to hIL8-77. D) Dose dependence of maximal level of Ca2+ response. E) Same as panels A, Cbut after exposure to hIL8-NL. F) Same as panel B but after exposure to hIL8-NL and –NS. The NL peptide was able to stimulate rapid Ca2+ increase whereas the NS peptide stimulated no Ca2+ increase. G) Same as panel E but for C-terminal peptide. H) Dose dependence of maximal level of Ca2+ response shown for all three hIL8 peptides. dF = F-Fo (Fo, average fluorescence intensity for each cell at rest; dF, change of fluorescence intensity for each cell upon ligand addition).

Similar experiments were performed with the hIL8 N- and C-terminal peptides (Fig. 1E-H ). We used NL-peptide (first 11aa of the 77aa form), NS-peptide (first 6aa of the 72aa form), and C-terminal 23 aa. hCXCR1 was activated by NL-peptide and Ca²⁺ increase was specific, as demonstrated by the lack of response in cells not subjected to Dox (Fig. 1E, F ). The NS-peptide, however, was unable to stimulate a Ca²⁺ response (Fig. 1F ), even at micromolar concentrations (Fig. 1H ). Treatment with hIL8 C-peptide at concentrations equivalent to those of the NL-peptide also resulted in activation of the receptor and an increase in intracellular Ca²⁺ (Fig. 1G, H ).

2. hIL8 N- and C-peptides stimulate MAP kinase activation
We analyzed ERK1/2 activation using immunoblot analysis and specific antibodies to the phosphorylated form of this kinase (see Fig. 3 of electronic version). Activation of this kinase depends on phosphorylation of Thr202 and Tyr204 of the protein and is completely inhibited by a specific inhibitor of MEK1. The results show that NL- and C-peptides both activate hCXCR1. We did not detect activation of either kinase by the NS-peptide, consistent with no increase detected in Ca²⁺ levels. At doses comparable to those used for Ca²⁺ mobilization, both peptides were able to stimulate phosphorylation of ERK1/2 in a Dox-dependent manner. Using signal transduction pathway inhibitors to explore the signaling requirement of ERK1/2 activation upon stimulation of hCXCR1, we found that PTX and HerbA significantly inhibited ERK1/2 phosphorylation induced by the peptides whereas PD98059 completely abrogated this activation. Inhibition of PKC did not significantly affect the ability of C-peptide and hIL8-72 to stimulate activation of ERK1/2, but inhibitors of this kinase were more effective in decreasing activation of ERK1/2 by the NL-peptide and IL8-77. These results suggest that signaling through hCXCR1 stimulated by IL8-72 and the C-peptide involves primarily PTX and src family kinase-sensitive pathways whereas signaling stimulated by IL8-77 and the NL-peptide activates additional pathways that involve other tyrosine kinases and PKC.

View larger version (31K): [in this window] [in a new window]   Figure 3. Summary of the functional activities of hIL8-72 and hIL-77 and their N and C termini. hCXCR1 responds to all ligands except the NS-peptide by stimulating intracellular Ca2+ increase, MAP kinase activation and chemotaxis of primary hMVEC, THP-1 monocytes, and THP-1-differentiated macrophages. Chemokines and their peptides also stimulate chemotaxis through hCXCR2. The NL-peptide is more efficient than the C terminus in stimulating chemotaxis through either receptor; it stimulates signals in part through PKC whereas the C-peptide does not.

These results show that the NL- and C-peptides of hIL8 are capable of activating hCXCR1, resulting in an intracellular Ca²⁺ increase and MAP kinase activation, whereas the NS-terminal peptide is able to activate the receptor. Furthermore, the presence of amino acids 1-6 in the N terminus appears to be important for PKC activation.

3. hIL8 N- and C-peptides stimulate chemotaxis in primary human cells
The results described above in NIH3T3 cells expressing hCXCR1 in a controlled manner show that the NL-peptide and the C-peptide of hIL8 (but not NS-peptide) can activate hCXCR1 and lead to signal transduction. To determine whether the peptides are able to activate primary cells in a biological assay, we used primary human microvascular endothelial cells (hMVEC) in chemotaxis assays. The functionality of the assay and the baseline for chemotaxis were established by using hIL8-77: we verified that hIL8-induced chemotaxis in these cells is specific and receptor dependent (Fig. 2 A). We then performed similar assays with the peptides and showed that only NL- and C-peptides stimulated migration of hMVEC in a dose-dependent manner (Fig. 2A ). Assays using the highest concentration of the peptide and antibodies specific for hCXCR1 or hCXCR2 showed that the effects of the peptides on chemotaxis of these cells are mediated by CXCR1 and/or CXCR2 (Fig. 2C ). Confirmation that the stimulated migration is chemotaxis is shown in Fig. 2B . We further tested that the peptides act through these receptors by performing competition assays during chemotaxis (Fig. 2D ). When cells were pretreated with excess peptides or excess hIL8, we found that subsequent application of hIL8 did not stimulate chemotaxis of hMVEC. These effects on hMVEC were also observed in THP-1 monocytes or THP-1-differentiated macrophages.

View larger version (34K): [in this window] [in a new window]   Figure 2. hIL8 N- and C-peptides stimulate chemotaxis in primary human microvascular endothelial cells. Cells were plated on the underside of the transwell unit at 1 x 105/100 µL, allowed to adhere, and the inserts inverted (to avoid any possible gravity effects) and placed inside the larger chamber. Potential chemoattractant agents were added to the upper chamber and cells were counted 4 h later. Effects of NL- and C-peptides of IL8 on hMVEC migration are dose dependent when agents are added to only the upper chamber (A) but abrogated when peptides are added to both chambers (B), thereby demonstrating chemotaxis. These effects are shown to be receptor mediated by inhibition with function-inhibiting antibodies (C) and by preincubation of cells with a high concentration of hIL8, NL-, or C-peptide, which resulted in a significant decrease in response to stimulation of the cultures by hIL8.

These results support and complement those performed to demonstrate Ca²⁺ release and MAP kinase activation. NL- and C-peptides of hIL8 are capable of stimulating hMVEC chemotaxis in a dose-dependent and hCXCR1/hCXCR2-mediated manner, whereas the NS-terminal peptide is not capable of eliciting chemotaxis of these cells, even at micromolar concentrations.

CONCLUSIONS AND SIGNIFICANCE

We conclude that the NL- and C-peptides of hIL8 activate both hCXCR1 and R2 independent of the core of the protein, leading to signal transduction mechanisms that result in biological functions such as chemotaxis (Fig. 3 ). It is not yet known where these peptides bind to the receptor, which amino acids are critical, or how they contribute to the specific biological processes implicated. It is important to determine where and when they are found in vivo.

These results enhance our knowledge of chemokine biology by identifying functional IL8-derived peptide ligands. Such identification should contribute to understanding how the multiple functions that chemokines exhibit under different physiological and pathological conditions can be achieved. Because these peptides are small, the findings could lead to the development of agonists or antagonists for modulation of function.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-1175fje; doi: 10.1096/fj.02-1175fje

² These authors contributed equally to this work.

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