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. 2001 Jun;103(2):244–254. doi: 10.1046/j.1365-2567.2001.01228.x

The pan-chemokine inhibitor NR58-3.14.3 abolishes tumour necrosis factor-α accumulation and leucocyte recruitment induced by lipopolysaccharide in vivo

Jill Reckless *, Lauren M Tatalick †,‡, David J Grainger *
PMCID: PMC1783230  PMID: 11412312

Abstract

Chemokines participate in the regulation of leucocyte recruitment in a wide variety of inflammatory processes, including host defence and diseases such as asthma, atherosclerosis and autoimmune disorders. We have previously described the properties of Peptide 3, the first broad-specificity chemokine inhibitor in vitro. Here, we report the properties of NR58-3.14.3, a retroinverso analogue of Peptide 3. NR58-3.14.3 inhibited leucocyte migration induced by a range of chemokines, including monocyte chemoattractant protein-1 (MCP-1) (2·5 nm), macrophage inflammatory protein-1α (MIP-1α) (5 nm), regulated on activation, normal T-cell expressed and presumably secreted (RANTES) (20 nm), stromal cell-derived factor-1α (SDF-1α) (25 nm) and interleukin-8 (IL-8) (30 nm), but did not affect migration induced by N-formyl-methionyl-leucyl-phenylalanine (FMLP) or complement C5a (> 100 µm). NR58-3.14.3 is therefore ≈1000-fold more potent than Peptide 3 but retains the broad-spectrum chemokine inhibitory activity of the parent peptide. In vivo, pretreatment with a systemic dose of 10 mg of NR58-3.14.3, but not the inactive derivative NR58-3.14.4, abolished leucocyte recruitment in response to intradermal injection of 500 ng of MCP-1 into rat skin. This suggests that NR58-3.14.3 is a functional chemokine inhibitor in vivo as well as in vitro. We utilized NR58-3.14.3 as a tool to investigate the role of chemokine activity during leucocyte recruitment in response to lipopolysaccharide (LPS) in vivo. NR58-3.14.3, but not NR58-3.14.4, abolished leucocyte recruitment in response to intradermal injection of 50 ng of LPS into rat skin. Furthermore, NR58-3.14.3 completely inhibited LPS-induced accumulation of tumour necrosis factor-α (TNF-α). This data is consistent with a model in which multiple chemokines act in parallel upstream of TNF-α. NR58-3.14.3 is therefore a powerful anti-inflammatory agent in vivo, suppressing proinflammatory cytokine production and leucocyte recruitment in response to endotoxin stimulus in rat skin.

Introduction

Recruitment of leucocytes to sites of inflammation is an important process in both physiological host defence and a range of common pathologies, including autoimmune disorders, atherosclerosis and sepsis.1–5 The signals that regulate leucocyte extravasation and accumulation at sites of inflammation are complex, being dependent on the co-ordinated expression of a wide range of proteins (including cell adhesion molecules and soluble signalling molecules) both by the inflamed tissue and by the leucocytes themselves.6–8

Leucocyte recruitment in response to injection of bacterial lipopolysaccharide (LPS) has been extensively studied as a model of inflammation.9–12 In the first hours following injection of LPS into the mouse peritoneum, levels of a wide range of proinflammatory cytokines are increased.12,13 In particular, circulating levels of interleukin-1 (IL-1), interleukin-6 (IL-6) and tumour necrosis factor-α (TNF-α) are strikingly elevated.9,12 Furthermore, inhibition of TNF-α activity (using either neutralizing antibodies or soluble TNF receptors) substantially reduces leucocyte recruitment in response to LPS and reduces mortality in response to higher doses of the LPS endotoxin.7,14–16

Chemokines are another large family of signalling molecules involved in the regulation of leucocyte trafficking.17,18 However, the role of different members of the chemokine family in orchestrating leucocyte recruitment in response to LPS is less well defined than for TNF-α. Early reports suggested that chemokines such as macrophage inflammatory protein-1α (MIP-1α) acted downstream of TNF-α as soluble TNF receptors blocked the induction of MIP-1α by LPS, but neutralizing antibodies to MIP-1α did not significantly affect TNF-α levels.10 According to this model, increased TNF-α activity induces MIP-1α, which in turn stimulates expression of adhesion molecules such as intracelluar adhesion molecule-1 (ICAM-1), which are thought to be essential for the leucocyte extravasation process. Recent data, however, has suggested that this simple model does not apply to all chemokines. For example, increased activity of monocyte chemoattractant protein-1 (MCP-1), a chemokine closely related to MIP-1α, may actually reduce inflammation, at least in some models of endotoxaemia. Zisman and colleagues12 reported that neutralizing antibodies to MCP-1 increased circulating levels of TNF-α and exacerbated subsequent LPS-induced mortality.

We have recently described a series of oligopeptides which act as functional chemokine inhibitors in vitro19 with broad specificity. The most potent of these peptides inhibited leucocyte migration induced by all of the chemokines tested – MCP-1, MIP-1α, regulated on activation, normal T-cell expressed and presumably secreted (RANTES), interleukin-8 (IL-8) and stromal cell-derived factor-1α (SDF-1α) – in the concentration range 2–10 µm, but had no effect on migration induced by non-chemokine ligands.19 Although there are now a range of reported low-molecular-weight antagonists specific for various chemokine receptors,20–24 these oligopeptides are the first pan-selective chemokine inhibitors to be described. The aim of the present study was to use these agents to test the effect of simultaneously inhibiting a range of chemokines on LPS-induced inflammation in vivo. Peptides are often unsuitable for use in vivo because of their short plasma half-life and susceptibility to proteolytic degradation. A range of strategies have been used in the past to convert peptides with biological activity in vitro into agents with activity in vivo, including the design and synthesis of retroinverso peptides in which l-amino acids are substituted with d-amino acids in the reverse sequence, generating a peptide mimetic with the amino acid side-chains in the same relative spatial orientation but with a backbone that is resistant to proteolytic degradation.25 Here we describe a cyclic retroinverso analogue of the peptide pan-selective chemokine inhibitor Peptide 3, termed NR58-3.14.3. This analogue retains the broad specificity of the peptide inhibitor, but is substantially more potent in the in vitro migration assays. We demonstrate that NR58-3.14.3 has powerful anti-inflammatory activity in vivo and use it as a tool to begin to dissect the relationship between chemokines and TNF-α in the regulation of leucocyte recruitment in response to LPS.

Materials and methods

Transwell migration assay using THP-1, a human myelomonocytic cell line

THP-1 cells (European Collection of Cell Cultures, Salisbury, Wilts., UK) were maintained at a density of 4 × 105−1 × 106 cells/ml in RPMI-1640 supplemented with 10% fetal calf serum (FCS) +20 µm 2-mercaptoethanol. THP-1 transwell migration assays were performed in 96-well disposable chemotaxis chambers fitted with a 5-µm polycarbonate filter (ChemoTx, Neuroprobe, Cabin John, MA), as described previously.19 Briefly, 29 µl of medium plus or minus chemoattractant was added to the lower compartment of each well. The framed filter was aligned with the holes in the corner of the filter frame and placed over the wells. THP-1 cells (5 × 104) in 25 µl of medium were added to the upper compartment of the transwell migration chamber. The serially diluted peptides to be tested were added with the THP-1 cells to the upper compartment of the chemotaxis chamber and incubated at 37° in a humidified atmosphere of 5% CO2 for 4 hr.

After incubation, any cells remaining in the upper compartment were removed and the membrane was incubated with 20 mm EDTA in phosphate-buffered saline (PBS). The number of cells that had migrated to the lower chamber was determined using the vital stain 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Sigma Chemical Co., Poole, UK). A standard curve consisting of a twofold dilution series of THP-1 cells (top standard 50 000 cells in 29 µl) was constructed. Migrated cells and cells in the wells for construction of the standard curve were stained by addition of 3 µl of MTT stock solution (5 mg/ml in RPMI-1640 without phenol red; Sigma Chemical Co.) and incubated at 37° for 4 hr. The media was carefully aspirated from each well, and the converted blue formazan dye was solubilized in 20 µl of dimethyl sulphoxide (DMSO). Absorbance of converted dye was measured at a wavelength of 595 nm using an enzyme-linked immunosorbent assay (ELISA) plate reader. The number of cells migrated in each well was determined by interpolation of the standard curve.

Transwell migration assay using human peripheral blood polymorphonuclear cells

Twenty-seven millilitres of fresh venous blood was taken and mixed with 3 ml of 3·8% trisodium citrate. After a 15-min incubation at room temperature, the anticoagulated blood was carefully layered over an equal volume of Polymorphprep (Nycomed, Majorstua, Oslo, Norway) and centrifuged at 498 g for 35 min using no brake, in accordance with the manufacturer's instructions. The purified polymorphonuclear cells (PMNC) (the lower of the two leucocyte bands) were removed carefully using a glass pipette, and mixed with an equal volume of 0·45% sodium chloride solution in order to restore normal osmolarity. The cells were centrifuged at 381 g for 10 min and the supernatant was removed. The cells were then washed three times with Dulbecco's A PBS before counting the PMNC and reconstituting the pellet to 1 × 107 cells/ml in Geys balanced salt solution (Sigma Chemical Co.) + 1 mg/ml of bovine serum albumin (BSA) (Sigma Chemical Co.). Transwell migration assays were performed in the same manner as described above for the THP-1 cells, except that 2·5 × 105 PMNC in 25 µl of Geys balanced salt solution +1 mg/ml of BSA were added to the upper compartment. When using the PMNC, the chamber was incubated at 37° in a humidified atmosphere of 5% CO2 for 1·5 hr. The number of PMNC that migrated were quantified by using the MTT vital stain, as described above.

Design and synthesis of NR58-3.14.3 and NR58-3.14.4

NR58-3.14.3 was synthesized by standard solid-phase peptide synthesis chemistry (Multiple Peptide Systems, San Diego, CA) using d-amino acids to yield the linear peptide H-cqiwkqkpdlc-NH2 (single-letter amino acid code), which was subsequently oxidatively cyclized between the terminal cysteine residues and purified by reverse-phase high-performance liquid chromatography (HPLC). The material used in these studies was > 99% pure, with a trifluoroacetate counter ion and a molecular weight (by mass spectrometry) of 1358.8. NR58-3.14.4 was synthesized from NR58-3.14.3 by a standard peptide-coupling reaction of d-alanine to the side-chain carboxyl group of the aspartic acid (D) residue of the cyclic retroinverso peptide, followed by purification over a reverse-phase column, as performed for NR58-3.14.3. The resultant material was > 99% pure with a trifluoroacetate counter ion and a molecular weight of 1428.7, as evaluated by mass spectrometry.

Design of the rat skin-injection study

Fifteen female Sprague-Dawley rats (200–250 g, 7–9 weeks old) (Charles River Labs, Wilmington, MA) were randomly allocated to three equal groups (n = 5). Each rat was acclimatized for a minimum of 2 days prior to the start of the study. Food and water were freely available ad libitum during the course of the experiment. Thirty minutes prior to intradermal injection of the proinflammatory stimulus, each animal was anaesthetized (by intraperitoneal injection at a dose of 0·165–0·5 ml/kg of body weight) with a mixture of 0·5 ml of ketamine (100 mg/ml vial), 0·1 ml of acepromazine (10 mg/ml), 0·25 ml of rompun (20 mg/ml) and 0·15 ml of sterile PBS (Bio-Whittaker, Wokingham, UK). Each rat was given a dorsal subcutaneous injection of sterile endotoxin-free PBS alone, or PBS containing 10 mg of the active compound (NR58-3.14.3) or the inactive control compound (NR58-3.14.4). Thirty minutes later, the following agents were injected at separate sites on the ventral abdomen of each rat: 500 ng of human recombinant carrier-free MCP-1 (R & D Systems, Abingdon, UK), 50 ng of LPS from Escherichia coli 0111:B4 (Sigma Chemical Co.) and sterile endotoxin-free PBS. All reagents, including MCP-1, were shown to be endotoxin-free by the limulus amebocyte lysate (LAL) method, as previously described.26

Animals were killed 24 hr later and the skin injection sites (MCP-1, LPS and PBS) were excised, washed in PBS and snap-frozen in OCT embedding medium. Tissue blocks were stored at −70° until sectioned.

Quantifying the cellular inflammatory response

For each of the three intradermal injection sites, 5-µm thick cryosections were collected through a total distance of 4 mm of rat skin. The sections were collected onto Poly-l-Lysine-coated slides (0·1%; Sigma Chemical Co.) and fixed in ice-cold acetone for 90 seconds, air dried and stored frozen at −20° until assayed. The sections were stained and analysed using the quantitative immunofluorescence protocol, as previously described.27 Five control sections and 10 test sections for each injection site were analysed using test sections equally spaced across the 4 mm of tissue that had been sectioned.

Briefly, each section was preblocked for 30 min at room temperature with a 3% solution of fatty acid-free BSA (Sigma Chemical Co.) in Tris-buffered saline (TBS), then incubated with the primary antibodies (described below) for 16 hr at 4° in a humidified box. Monocytes and macrophages were detected using an anti-rat CD14 antibody (clone ED2; Serotec, Kidlington, UK) at a concentration of 3·125 µg/ml. B and T lymphocytes were stained using anti-rat B cells (clone RLN9D3; Serotec) and anti-rat T cells (clone MRC OX-52; Serotec) at a concentration of 10 µg/ml and 878 µg/ml, respectively. Neutrophils were detected using an anti-rat granulocyte antibody (clone HIS48; Serotec) at a 1:10 dilution of the tissue culture supernatant. In addition, inflammatory proteins were also measured using the following antibodies: goat polyclonal anti-JE (R & D Systems; AB-479-NA; JE is the mouse homologue of human MCP-1), anti-mouse TNF-α antibody (R & D Systems; AB-410-NA), and anti-human IL-8 antibody (R & D Systems; AB-208-NA), all used at a concentration of 50 µg/ml. After 16 hr of incubation with primary antibody, the sections were washed three times for 3 min with PBS, and were then incubated with an appropriate secondary antibody for 6 hr at room temperature. Fluorescein isothiocyanate (FITC)- and rhodamine (TRITC)-conjugated affinipure anti-mouse, rat or goat immunoglobulin Gs (IgGs) (with minimal cross-reactivity) were used as the secondary antibodies at ≈30 µg/ml (Jackson Immuno Research, West Grove, PA). The sections were then washed as described above and allowed to air dry, then coverslipped using Citifluor AF-1 (Agar Scientific, Stansted, UK).

Data and statistical analysis

Three images from each section were randomly selected and captured using a fluorescence microscope (AX70; Olympus, Southall, UK) attached to a CCD camera (Hamamatsu, Welwyn Garden City, UK), as described previously27 (a total of 15 control images and 30 test images per injection site were obtained for each of the different primary antibody stains). The images were subsequently analysed using customised software (Openlab; Improvision, UK) and NIH Image. The total tissue area in each image was determined by density-slicing the image, and the area of tissue stained was then obtained by performing a second density-slice operation. The percentage of tissue area, which was stained using each of the primary antibodies, was then calculated. Because the staining with most of the primary antibodies used showed high spatial variability across the injection site, as a result of the focal nature of the inflammatory response, we reported the median values of the 30 test images taken at each injection site. In all cases the appropriate control values were subtracted from the test values, in accordance with the provision for quantitative immunofluorescence set out by Mosedale and colleagues.27 The three groups were then compared using the non-parametric Kruskal–Wallis test, with a P-value of < 0·05 taken to indicate significance.

Results

Structure of NR58-3.14.3 and NR58-3.14.4

As an approach to developing analogues of Peptide 3 able to function as broad-spectrum chemokine inhibitors in vivo, we adopted a similar strategy to Jameson and colleagues, who generated biologically active retroinverso analogues of their CD4 fragment.25 We selected the most potent variant of the Peptide 3 sequence reported previously (Leu4Ile11-peptide 3)19 and added an additional terminal cysteine residue at position 13 to allow oxidative cyclization through the two thiol groups. This retroinverso peptide analogue was then synthesized by standard solid-phase peptide synthesis chemistry and was termed NR58-3.14.3 (Fig. 1).

Figure 1.

Figure 1

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Schematic representation of NR58-3.14.3 and NR58-3.14.4. The retroinverso analogue of Leu4Ile11Cys13-Peptide 3 is shown in the left panel. The N- and C termini are indicated, with shaded circles representing the α-carbon atoms of the 13 amino acids with d configuration. The retroinverso peptide is oxidatively cyclized between the terminal cysteine side-chains. The right panel shows the derivative NR58-3.14.4, which differs from NR58-3.14.3 by the addition of d-alanine linked to the side-chain of the aspartic acid through an isopeptide bond (shaded box).

A second retroinverso peptide analogue of Peptide 3, termed NR58-3.14.4, was also synthesized, with a d-alanine linked to the aspartic acid residue through an iso-peptide bond (as shown in the shaded box in Fig. 1). Although NR58-3.14.4 has a very similar composition to NR58-3.14.3, it has little or no activity as a chemokine inhibitor (see below) and was therefore used throughout this study as an inactive control.

In vitro properties of NR58-3.14.3 and NR58-3.14.4

We first tested whether NR58-3.14.3 shared the ability to inhibit migration induced by the range of chemokines that was described previously for Peptide 3.19 For each chemokine, we first determined the concentration in the lower compartment of the migration chamber that induced a maximal migratory response. For example, raising the concentrations of MCP-1 (filled squares) and SDF-1α (filled triangles) first increased the number of cells migrating, then at higher concentrations showed decreasing promigratory activity (Fig. 2a). This ‘bell-shaped’ dose–response curve is typical of agents, such as chemokines, that are purely chemotactic (as opposed to chemokinetic). In subsequent assays, the dose of chemokine was used that elicited the maximal promigratory effect.

Figure 2.

Figure 2

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Effect of NR58-3.14.3 on chemokine-induced migration in vitro. (a) Effect of various doses of the chemokines monocyte chemoattractant protein-1 (MCP-1) (filled squares) and stromal cell-derived factor-1α (SDF-1α) (filled triangles) or of NR58-3.14.3 (closed circles) or NR58-3.14.4 (open circles), in the lower chamber of the assay plate on migration of THP-1 cells (see the Materials and methods). Similar experiments were performed to determine the dose of each chemokine that elicited a maximal migratory response. (b), (c), (d), (e), (f) Effect of different concentrations of NR58-3.14.3 (filled circles) or NR58-3.14.4 (open circles) on migration of THP-1 cells induced by MCP-1 (50 ng/ml), macrophage inflammatory protein-1α (MIP-1α) (3·125 ng/ml), regulated on activation, normal T-cell expressed and presumably secreted (RANTES) (100 ng/ml), SDF-1α (75 ng/ml) and N-formyl-methionyl-leucyl-phenylalanine (FMLP) (100 nm). (g), (h) Effect of various concentrations of NR58-3.14.3 on migration of freshly prepared human peripheral blood polymorphonuclear cells (PMNC) induced by interleukin-8 (IL-8) (100 ng/ml) or complement C5a (50 nm). Each value represents the mean ± standard error of the mean (SEM) of triplicate determinations and each panel is representative of three separate experiments.

The retroinverso peptide analogues were incubated with the THP-1 cells in the upper compartment of the migration chamber, with the various chemokines added to the lower chamber. After allowing the cells to migrate for 4 hr, the number of cells that had migrated were quantified using the vital stain MTT. NR58-3.14.3 potently inhibited THP-1 migration induced by MCP-1 (Fig. 2b) with a 50% effective dose (ED50) of 2·5 nm. In contrast, NR58-3.14.4 was significantly less active as an inhibitor of MCP-1-induced migration, failing to inhibit migration by 50%, even at 100 µm. As a result, in our in vivo studies we used NR58-3.14.4 as an inactive control of very similar composition to the active NR58-3.14.3.

NR58-3.14.3 might reduce MCP-1-induced migration in this assay system, either by inhibiting MCP-1 function or by providing an independent promigratory signal of opposite direction. Two equal and opposing gradients of promigratory factors might result in no significant directed migration of the cells. To investigate this possibility, we tested whether NR58-3.14.3 placed only in the lower chamber of the migration assay (in place of the chemokine) could induce migration. However, neither NR58-3.14.3 (filled circles) nor NR58-3.14.4 (open circles) in the concentration range of 1 fM to 1 mm were able to induce any THP-1 migration whatsoever (Fig. 2a). We conclude therefore that NR58-3.14.3 probably acts as an inhibitor of MCP-1 function.

NR58-3.14.3 also inhibited migration of THP-1 cells induced by a range of other chemokines, including MIP-1α, RANTES and SDF-1α (Fig. 2c, 2d, 2e). In each case, NR58-3.14.3 was a potent inhibitor with an ED50 in the range of 5–25 nm, and in each case inhibited migration by > 95% at 100 µm (P < 0·0001 in each case). However, in marked contrast to the effect of NR58-3.14.3 on CC and CXC chemokine-induced migration, NR58-3.14.3 had no significant effect on FMLP-induced migration, even at 100 µm (Fig. 2f).

We previously tested the effect of Peptide 3 on IL-8-induced migration of THP-1 cells.19 However, we found that IL-8 at all concentrations tested was only a very weak chemotactic agent for THP-1 cells. In order to better examine the effect of NR58-3.14.3 on IL-8-induced migration, we therefore used freshly prepared human peripheral blood PMNCs in the migration assay. IL-8 is a powerful chemoattractant for human PMNCs, causing more than 60% of the cells added to the upper chamber to migrate in response to 100 ng/ml of IL-8. NR58-3.14.3 inhibited IL-8-induced migration of PMNCs from three separate donors with an ED50 of ≈30 nm (Fig. 2g).

To determine whether NR58-3.14.3, like Peptide 3, inhibited chemokine-induced migration but not neutrophil migration induced by other factors, we tested the effect of NR58-3.14.3 on migration induced by complement C5a. C5a (50 nm) induced migration of human PMNCs to similar extent as IL-8 (≈ 40% of the total cells placed in the upper compartment of the well underwent migration). However, in marked contrast to the effect of NR58-3.14.3 on IL-8-induced migration, NR58-3.14.3 had no significant effect on C5a-induced migration, even at 100 µm (Fig. 2h).

Taken together, these results demonstrate that NR58-3.14.3 is a broad-specificity functional inhibitor of chemokine-induced migration, but has no effect on migration induced by other agents. NR58-3.14.3 therefore has very similar properties to Peptide 3, although it is 100–1000-fold more potent in vitro.

Effect of NR58-3.14.3 on MCP-1-induced inflammation in vivo

We have shown that NR58-3.14.3 inhibits THP-1 migration induced by a range of chemokines in a transwell migration assay in vitro. Next, we tested whether NR58-3.14.3 was able to inhibit leucocyte recruitment in vivo. As a proof-of-concept study we used a rat skin inflammation model in which MCP-1 was injected intradermally into the ventral abdomen of a rat to induce cellular inflammation. Thirty minutes prior to MCP-1 administration, each animal was randomly allocated into three groups (n = 5) and treated with 10 mg of NR58-3.14.3, 10 mg of the control inactive compound (NR58-3.14.4), or PBS vehicle, by subcutaneous injection. Twenty-four hours later, the animals were killed, and the skin around each intradermal injection site was excised, embedded in OCT embedding medium and frozen at −70°. The extent and composition of the inflammatory response was then measured using antibodies specific for the different leucocyte subsets (as described in the Materials and methods) using quantitative immunofluorescence techniques.

As expected, the rats which received PBS vehicle pretreatment had a small but readily detectable number of leucocytes patrolling the uninflamed region of skin (intradermally injected with PBS). The majority of leucocytes present were monocyte/macrophages, with a small number of B and T lymphocytes and an occasional neutrophil (Fig. 3). However, at the site, which received an intradermal injection of MCP-1, there was a significant inflammatory response (Fig. 3). The majority of recruited leucocytes were CD14+ cells (≈ 30–40% of the inflammatory infiltrate were monocyte/macrophages), but the numbers of B and T cells were also significantly increased (Fig. 3). In marked contrast, there was no significant increase in the number of neutrophils at the MCP-1 injection site compared to the PBS-vehicle site (< 10% increase, P > 0·05, Fig. 3). This is consistent with a number of previous studies, which reported a mononuclear cell-rich inflammatory response elicited by MCP-1 in vivo.28–30

Figure 3.

Figure 3

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Effect of NR58-3.14.3 and NR58-3.14.4 on leucocyte recruitment in response to monocyte chemoattractant protein-1 (MCP-1) in vivo. The number of neutrophils, monocyte/macrophages and B and T lymphocytes, estimated from the quantitative immunofluorescence staining of 10 separate sections across 4 mm of dermis surrounding either the 500-ng MCP-1 or phosphate-buffered saline (PBS) vehicle injection sites, are shown for each of five rats in each group. The three groups were pretreated with PBS vehicle alone, or with 10 mg of NR58-3.14.4 or 10 mg of NR58-3.14.3. The bar represents the median staining for each leucocyte type in each group of animals, and the filled circles show the individual values for the five animals. Where the group of animals pretreated with either NR58-3.14.4 or NR58-3.14.3 differed from those pretreated with vehicle alone, the P-value (Mann–Whitney U-test) is shown.

Pretreatment of the animals with the control inactive agent, NR58-3.14.4, did not affect the number of any of the leucocyte subtypes at either the PBS or MCP-1 injection site (< 10%, P > 0·05, Fig. 3), compared with animals pretreated with PBS vehicle. In contrast, all the MCP-1-induced leucocyte recruitment was abolished in animals pretreated with 10 mg of NR58-3·14·3 (Fig. 3). There was an approximate sevenfold reduction in the number of monocyte/macrophages at the MCP-1 injection site compared to animals pretreated with either PBS or NR58-3.14.4 (P < 0·01, Fig. 3). Pretreatment with NR58-3.14.3 also reduced, to a similar extent, the number of B and T cells recruited in response to MCP-1 (P < 0·01, Fig. 3). In all animals pretreated with NR58-3.14.3, the number of leucocytes present at the MCP-1 injection site was less than the number of leucocytes patrolling the uninflamed dermis at the PBS injection site (Fig. 3). Systemic pretreatment with NR58-3.14.3 completely abolished the inflammatory response induced by intradermal injection of MCP-1.

To begin to investigate the mechanism of the anti-inflammatory effect of NR58-3.14.3, we also measured the levels of a number of proinflammatory cytokines in the rat dermis, using quantitative immunofluorescence. There was a low, but detectable, level of TNF-α at the PBS injection site, which increased approximately twofold after MCP-1 injection (P < 0·01; Fig. 4, Fig. 5g). Pretreatment with NR58-3.14.3, but not with the inactive NR58-3.14.4 or PBS vehicle, completely abolished TNF-α staining at both the MCP-1 and PBS injection sites (P < 0·01, Fig. 4).

Figure 4.

Figure 4

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Effect of NR58-3.14.3 and NR58-3.14.4 on proinflammatory cytokine accumulation in response to monocyte chemoattractant protein-1 (MCP-1) in vivo. The levels of tumour necrosis factor-α (TNF-α) and endogenous rat MCP-1, estimated from the quantitative immunofluorescence staining of 10 separate sections across 4 mm of dermis surrounding either the MCP-1 or the phosphate-buffered saline (PBS) vehicle injection sites, are shown for each of five rats in each group. The three groups contained the same animals as in Figure 3. The bar represents the median staining for each leucocyte type in each group of animals, and the filled circles show the individual values for the five animals. Where the group of animals pretreated with either NR58-3.14.4 or NR58-3.14.3 differed from those pretreated with vehicle alone, the P-value (Mann–Whitney U-test) is shown.

Figure 5.

Figure 5

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Immunofluorescence staining for leucocytes and proinflammatory cytokines at the monocyte chemoattractant protein-1 (MCP-1) injection site of animals pretreated with vehicle alone. Representative images of serial sections stained for nuclei (b), neutrophils (c), monocyte/macrophages (d), B lymphocytes (e), T lymphocytes (f), tumour necrosis factor-α (TNF-α) (g) and endogenous rat MCP-1 (h), were compared with a control section stained with labelled second antibody only (a). Note that a white arrow denotes the skin surface in (a). Bar represents 50 µm.

We also examined the level of endogenous MCP-1 antigen at these injection sites, using an antibody that is highly specific for the endogenous rat MCP-1 compared with the injected human MCP-1. As with TNF-α, there was an approximate twofold increase in staining for endogenous MCP-1 at the MCP-1 injection site compared to the PBS vehicle site, but this did not reach statistical significance (P > 0·05; Fig. 4, Fig. 5h). We observed that there was significantly greater variation between animals in the dermal level of endogenous MCP-1 at both the PBS and MCP-1 injection sites than for the other antigens examined. Pretreatment with NR58-3.14.3 significantly reduced the levels of endogenous MCP-1 at both the PBS and MCP-1 injection sites, while pretreatment with NR58-3.14.4 or vehicle had no effect (P < 0·05; Fig. 4).

We conclude from these experiments that NR58-3.14.3 inhibits leucocyte recruitment and the associated increase in proinflammatory cytokine expression in response to MCP-1 injected into rat dermis. This suggests that NR58-3.14.3 acts as a functional chemokine inhibitor in vivo as well as in vitro.

Effect of NR58-3.14.3 on LPS-induced inflammation in vivo

Bacterial LPS induces a massive inflammatory response, in which a range of proinflammatory cytokines orchestrate the recruitment of neutrophils, lymphocytes and monocyte/macrophages.9–13 However, it remains unclear what role chemokines play in the leucocyte recruitment in response to bacterial LPS in vivo. We have therefore utilized NR58-3.14.3, the first broad-spectrum chemokine inhibitor to be described, to determine the effect of inhibiting multiple chemokines during LPS-induced inflammation.

We used the same model of dermal inflammation described above, in which LPS was injected intradermally 30 min after pretreatment with either 10 mg of NR58-3.14.3, 10 mg of NR58-3.14.4 or vehicle alone. As expected, LPS induced a strong inflammatory response, with a large increase in all the leucocyte subtypes examined (Fig. 6, Fig. 7). In marked contrast to the effect of intradermal injection of MCP-1, LPS induced a strong neutrophil inflammatory response (≈ 15-fold increase compared with the PBS injection site, P < 0·01, Fig. 6). Pretreatment with NR58-3.14.3, but not with NR58-3.14.4 or vehicle, completely abolished leucocyte recruitment in response to LPS injection (P < 0·01 for each leucocyte subset; Fig. 6), and the number of leucocytes present in the dermis was reduced back to, or below, the levels in uninflamed dermis at the PBS injection site.

Figure 6.

Figure 6

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Effect of NR58-3.14.3 and NR58-3.14.4 on leucocyte recruitment in response to lipopolysaccharide (LPS) in vivo. The number of neutrophils, monocyte/macrophages, and B and T lymphocytes, estimated from the quantitative immunofluorescence staining of 10 separate sections across 4 mm of dermis surrounding either the 50-ng LPS or the phosphate-buffered saline (PBS) vehicle injection sites, are shown for each of five rats in each group. The three groups were pretreated with PBS vehicle alone, or with 10 mg of NR58-3.14.4 or 10 mg of NR58-3.14.3. The bar shows the median staining for each leucocyte type in each group of animals, and the filled circles represent the individual values for the five animals. Where the group of animals pretreated with either NR58-3.14.4 or NR58-3.14.3 differed from those pretreated with vehicle alone, the P-value (Mann–Whitney U-test) is shown.

Figure 7.

Figure 7

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Immunofluorescence staining for leucocytes and proinflammatory cytokines at the lipopolysaccharide (LPS) injection site of animals pretreated with vehicle alone. Representative images of serial sections stained for nuclei (b), neutrophils (c), monocyte/macrophages (d), B lymphocytes (e), T lymphocytes (f), tumour necrosis factor-α (TNF-α) (g), monocyte chemoattractant protein-1 (MCP-1) (h) and interleukin-8 (IL-8) (i), were compared with a control section stained with labelled second antibody only (a). Note that a white arrow denotes the skin surface in (a). Bar represents 50 µm.

To further investigate the role of chemokines in LPS-induced inflammation, we measured the levels of endogenous MCP-1, IL-8 and TNF-α in the rat dermis. Intradermal injection of LPS, like MCP-1, increased the levels of TNF-α in the tissue by approximately twofold (P < 0·05; Fig. 8). Pretreatment with NR58-3.14.3, but not with NR58-3.14.4 or vehicle, completely inhibited the LPS-induced increase of TNF-α.

Figure 8.

Figure 8

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Effect of NR58-3.14.3 and NR58-3.14.4 on proinflammatory cytokine accumulation in response to lipopolysaccharide (LPS) in vivo. The levels of tumour necrosis factor-α (TNF-α), monocyte chemoattractant protein-1 (MCP-1) and interleukin-8 (IL-8), estimated from the quantitative immunofluorescence staining of 10 separate sections across 4 mm of dermis surrounding either the LPS or phosphate-buffered saline (PBS) vehicle injection sites, are shown for each of five rats in each group. The three groups contain the same animals as in Figure 6. The bar represents the median staining for each leucocyte type in each group of animals, and the filled circles show the individual values for the five animals. Where the group of animals pretreated with either NR58-3.14.4 or NR58-3.14.3 differed from those pretreated with vehicle alone, the P-value (Mann–Whitney U-test) is shown.

As with TNF-α, levels of both endogenous MCP-1 and IL-8 in the dermis were significantly increased following LPS injection (a two- to threefold increase was observed in both cases, P < 0·05, Fig. 7h, Fig. 7i, Fig. 8). NR58-3.14.3, but not PBS vehicle, significantly reduced the levels of both these chemokines in rat dermis to, or below, the levels present in uninflamed skin at the PBS injection site (P < 0·01 and P < 0·05 for MCP-1 and IL-8, respectively, Fig. 8). NR58-3.14.4 had no effect on the level of endogenous MCP-1 or the level of IL-8 staining at the LPS site. In summary, pretreatment with the broad-specificity chemokine inhibitor, NR58-3.14.3, completely inhibited the inflammatory response induced by LPS in rat skin in vivo.

Discussion

Chemokines have been implicated in a range of inflammatory processes, both in normal host defence and in various pathologies.31–33 As a result, there have been a number of reports of chemokine inhibitors.20–24,34,35 The first inhibitors to be described were chemokine proteins with modifications to the N-terminal region, such as Met-RANTES and AOP-RANTES.34,35 More recently, a large number of small molecule antagonists of many different chemokine receptors, including CCR2, CCR5 and CXCR1, have been reported.20–24 These chemokine inhibitors act as classical receptor antagonists, blocking the interaction of chemokine ligand with the receptor and are usually specific for one or two closely related receptors. In marked contrast, we have previously described a peptide sequence from human MCP-1, which was a broad-specificity chemokine inhibitor with potency in the low micromolar range.19 This peptide, termed Peptide 3, was the first chemokine inhibitor to be reported that simultaneously inhibited both CC and CXC chemokines.19

Here we describe a retroinverso peptide analogue of Peptide 3, which retains the key biological properties of Peptide 3 in vitro. This analogue, NR58-3.14.3, inhibited leucocyte migration induced by MCP-1, MIP-1α, RANTES, IL-8 and SDF-1α, but did not affect migration induced by FMLP or complement C5a (Fig. 2). However, NR58-3.14.3 was ≈1000-fold more potent than the parent peptide (inhibiting MCP-1-induced migration with an ED50 of 2·5 nm), suggesting that it may be a useful broad-specificity chemokine inhibitor in vivo. At present, the molecular mechanisms responsible for the functional inhibition of chemokine-induced migration by NR58-3.14.3, or indeed Peptide 3, remain unknown. However, NR58-3.14.3 does not block binding of radiolabelled chemokines to their receptors (data not shown), suggesting that it is not acting as a receptor antagonist with broad specificity. In addition, NR58-3.14.3 does not promote receptor internalization (which is the probable mechanism of action of N-terminally modified chemokines such as AOP-RANTES). Instead, it is conceivable that NR58-3.14.3 blocks intracellular signals necessary for migration, which are generated by the chemokine receptors but not by other G-protein coupled receptors (GPCRs), such as the C5a or FMLP receptors.

As a first test of the ability of NR58-3.14.3 to block chemokine function in vivo, we examined its effect on leucocyte recruitment in response to intradermal injection of recombinant human MCP-1. The first study of intradermal injection of MCP-1 found only a mild inflammatory response following injection of 1 µg of protein into mouse footpad.36 However, several more recent studies reported a more striking inflammatory response to quantities of MCP-1 as low as 50 ng injected into rat skin in vivo.28–30 Consistent with these reports, we found a mononuclear cell-rich inflammatory response following injection of 500 ng of MCP-1, raising the possibility that the magnitude of the response to MCP-1 varies with the injection site. It is important to note that injection of MCP-1 did not increase the number of PMNC in the dermis at the injection site. This confirms that the recombinant chemokine preparation we used was essentially free of endotoxin (which promotes inflammation with a strong neutrophil component), indicating that the observed inflammatory response is directly the result of chemokine function. As pretreatment with 10 mg of NR58-3.14.3, but not its inactive analogue NR58-3.14.4, completely abolished leucocyte recruitment in response to MCP-1, we concluded that NR58-3.14.3 is a functional chemokine inhibitor in vivo as well as in vitro (Fig. 2, Fig. 3).

The present study is representative of three similar studies, all of which show a statistically significant reduction in leucocyte recruitment induced by either MCP-1 or LPS (P < 0·05 in every case), when animals were pretreated with NR58-3.14.3. In one of these studies, rats were pretreated with three different doses (100 µg, 1 mg or 10 mg) of NR58-3.14.3 prior to the intradermal injection of MCP-1, LPS or PBS vehicle. There was a dose-dependent decrease in the number of CD14+ cells and B cells (data not shown), maximal at 10 mg, which was the dose used for the complete analysis presented in the present study. NR58-3.14.3 is, however, very non-toxic (the acute 50% lethal dose [LD50] in mice is > 500 mg when administered via the intravenous route; data not shown), suggesting that NR58-3.14.3 has a wide therapeutic index. The relatively low potency of NR58-3.14.3 in vivo (showing maximal effect at a dose of 10 mg), compared with its potency in vitro (an ED50 of 2·5–25 nm) probably results from the rapid clearance of the compound from plasma. NR58-3.14.3 has a plasma half-life of less than 30 min following intravenous injection into mice,37 largely as a result of renal clearance.

A broad-specificity chemokine inhibitor, such as NR58-3.14.3, is a useful new tool for investigating the network of proinflammatory cytokines responsible for orchestrating leucocyte recruitment in vivo. For example, injection of the endotoxin LPS into rat dermis leads to a massive inflammatory response rich in both polymorphonuclear and mononuclear leucocytes.9–12 A large number of reports suggest that TNF-α plays a pivotal role in driving leucocyte recruitment in this model, as neutralization of TNF-α activity (either using antibodies or soluble TNF receptors) reduces leucocyte accumulation and promotes survival in lethal endotoxaemia models.7,14–16 The role of chemokines in this process is less clear: neutralization of MIP-1α reduces LPS-induced inflammation,10 but neutralization of MCP-1 increases it.12 Standiford et al. suggested that chemokines acted downstream of TNF-α, as TNF-α blockade reduced MIP-1α accumulation but neutralization of MIP-1α did not affect TNF-α levels. Our studies with NR58-3.14.3 suggest a different explanation: that simultaneous inhibition of the function of multiple chemokines completely abolished TNF-α up-regulation and subsequent leucocyte recruitment. This would suggest that chemokine activity is necessary for the earliest leucocyte recruitment and TNF-α production that occurs in response to LPS.

Based on the observations of this study, we propose that LPS induces production of multiple chemokines by the cells already resident in the dermis. In response to these chemokines, the first inflammatory cells are recruited and begin to synthesize TNF-α, which rapidly magnifies the inflammatory lesion. These early leucocytes also synthesize further chemokines, which participate in the feedback loop responsible for recruiting the bulk of the inflammatory cells to the injection site. Thus, blockade of TNF-α dramatically reduces the size of the inflammatory infiltrate by preventing secondary recruitment, but neutralizing antibodies to individual chemokines cannot prevent TNF-α up-regulation by the first leucocytes to be recruited because this early recruitment is initiated by several chemokines. In this model, multiple chemokines acting in parallel lie upstream of TNF-α. It is, however, impossible to distinguish whether NR58-3.14.3 indirectly decreased TNF-α production by inhibiting leucocyte recruitment (as leucocytes are a major source of TNF-α) or whether NR58-3.14.3 directly inhibited TNF-α production, by either keratinocytes or leucocytes, and hence blocked leucocyte recruitment.

Nevertheless, irrespective of the molecular pathways involved, it is already clear that NR58-3.14.3 is a powerful inhibitor of acute inflammatory responses in vivo (Figs 3–8). It will be interesting to determine whether NR58-3.14.3 can prevent inflammation in animal models more relevant to human inflammatory diseases. Equally importantly, it should be possible to use NR58-3.14.3 to determine whether the chemokines it is able to inhibit have essential roles in normal physiology. For example, it is possible that chronic treatment with a chemokine inhibitor will lead to immunosuppression and compromise of the natural host defences. However, despite considerable effort it has not yet been possible to demonstrate physiological differences between individuals who are homozygous for an inactivating mutation in the chemokine receptor CCR5 (CCR5Δ32) and wild-type individuals, suggesting that there may be considerable redundancy within the chemokine system. Much further work will be necessary to determine whether molecules such as NR58-3.14.3 might have clinically useful anti-inflammatory properties in humans.

Abbreviations

FMLP

N-formyl-methionyl-leucyl-phenylalanine

HPLC

high-performance liquid chromatography

IL-8

interleukin-8

LPS

lipopolysaccharide

MCP-1

monocyte chemoattractant protein-1

MIP-1α

macrophage inflammatory protein-1α

MTT

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

RANTES

regulated on activation, normal T-cell expressed and presumably secreted

SDF-1α

stromal cell-derived factor-1α

TNF-α

tumour necrosis factor-α

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