Therapeutic efficacy and immunological response of CCL5 antagonists in models of contact skin reaction

Abstract

Skin-infiltrating T-cells play a predominant role in allergic and inflammatory skin diseases such as atopic dermatitis, psoriasis and allergic contact dermatitis. These T-cells are attracted by several chemotactic factors including the chemokine CCL5/RANTES, a CC chemokine inducing both the migration and activation of specific leukocyte subsets. CCL5 has been found to be associated with various cell-mediated hypersensitive disorders such as psoriasis, atopic dermatitis and irritant contact dermatitis. We have used two antagonists, the first, Met-CCL5, a dual CCR1/CCR5 antagonist and the second, a variant in which GAG binding is abrogated, (44)AANA(47)-CCL5, which acts as a dominant negative inhibitor of CCL5. The antagonists were tested in two models of contact skin reaction. The first, irritant contact dermatitis (ICD) is a pathological non-specific inflammatory skin condition arising from the release of pro-inflammatory cytokines by keratinocytes in response to haptens, usually chemicals. The second, contact hypersensitivity (CHS) is a T-cell dependent model, mimicking in part the T-cell-mediated skin diseases such as psoriasis. In both models, the CCL5 antagonists showed therapeutic efficacy by reducing swelling by 50% as well as the reduction of soluble mediators in homogenates derived from challenged ears. These results demonstrate that blocking the receptor or the ligand are both effective strategies to inhibit skin inflammation.

Figure 1. Therapeutic efficacy of [⁴⁴AANA⁴⁷]-CCL5 and Met-CCL5 on ear swelling.

Ear Swelling in head to head Oxazolone-induced CHS. CHS was induced by sensitization on shaved back with 3% Oxazolone at day 0. Challenge with 0.5% Oxazolone was done at day 5 to the right ear of sensitized mice. Mice were treated 30′ after irritation once with CCL5 antagonists at 0.5 mg/kg ip. Dexamethasone 10 mg/kg was used as reference compound. Results are given as mean ± SEM of n = 8 mice per group. All statistical analyses were performed using two-way ANOVA followed by Bonferroni's post test (***p<0.001 vs vehicle for each time point- 24,48,72 h).

Figure 2. Therapeutic efficacy of [⁴⁴AANA⁴⁷]-CCL5 and Met-CCL5 on ear swelling, using different mouse models of contact skin reaction.

A) represents [⁴⁴AANA⁴⁷]-CCL5 and B) Met-CCL5 therapeutic effect respectively on swelling in Oxazolone-induced CHS mouse model at 0.05–0.5–1 mg/kg dose via i.p. Dexamethasone 10 mg/kg sub-cute (s.c.) was used as reference compound. C) represents [⁴⁴AANA⁴⁷]-CCL5 and D) Met-CCL5 therapeutic effect respectively on swelling in CHS mouse model using DNFB as hapten at 0.05–0.5–1 mg/kg via i.p. Dexamethasone 0.5 mg/kg s.c. was used as reference compound. E–F) are showing therapeutic efficacy of [⁴⁴AANA⁴⁷]-CCL5 and Met-CCL5 in ICD mouse model tested at 0.5–1–5 mg/kg and 0.05–0.1–0.5 mg/kg i.p respectively. Dexamethasone 0.5 mg/kg was used as reference compound. All data were expressed as mean ± SEM of n = 8/group. All statistical analyses were performed using one-way Anova followed by Dunnett's multiple comparison post test (*p<0.05, **p<0.01, ***p<0.001 vs saline-treated vehicle group).

Figure 3. Myeloperoxidase activity in ICD and CHS ear extracts.

Determination of MPO was used as an indirect measure of neutrophils recruitment and content of ear tissue. Myeloperoxidase activity was tested respectively in ICD and CHS mouse models. In ICD mouse model, one biopsy of challenged ear (5 mm of diameter) of balb/c 8–12 weeks females was irritated with Croton oil and treated after irritation with A) [⁴⁴AANA⁴⁷]-CCL5 at 0.5–1–5 mg/kg and B) Met-CCL5 at 0.025–0.05–0.1–0.5 mg/kg via intraperitoneal injection. In DNFB-induced CHS (Fig. C/D) one biopsy of challenged ear (5 mm of diameter) of balb/c 8–12 weeks females was challenged by 0.2% DNFB and treated 30′ after irritation with [⁴⁴AANA⁴⁷]-CCL5 and Met-CCL5 at 0.05–0.5–0.1 mg/kg via intra-peritoneal injection. Results are given in % of vehicle for comparison of therapeutic efficacy of the two mutants, as mean ± SEM of n = 4 mice.

Figure 4. Cytokines and Chemokines profile in ICD and CHS ear extract.

A) Cytokine and Chemokine profiles in ICD mouse model. CBA assay was performed on ear extracts of Balb/c female 8–12 weeks of age. Panel 1: Treatments used are represented by [⁴⁴AANA⁴⁷]- CCL5 at 0.5–1–5 mg/kg ip. [⁴⁴AANA⁴⁷]- CCL5 at 5 mg/kg was able to reduce significantly levels of IL-12p70 (*p<0.05), IL-6 (**p<0.01) and MCP-1 (**p<0.01). A trend to reduction has been observed for IL-6 and MCP-1 at 1 mg/kg dose. Panel 2: Treatments used are represented by Met-CCL5 at 0.05–0.1–0.5 ip. Met-CCL5 at 0.05–0.1–0.5 mg/kg was able to significantly reduce IL-6 (**p<0.01) and MCP-1 (**p<0.01, *p<0.05). A trend to reduction is shown for IL-12p70. Results are given as mean ± SEM of n = 4 mice per group. All statistical analyses were performed using one-way ANOVA followed by Dunnett's multiple comparison test (*p<0.05, **p<0.01 vs vehicle). B) Cytokine and Chemokine profiles in DNFB-induced CHS. CBA assay was performed on ear extracts of sensitized and challenged Balb/c female 8–12 weeks of age. Panel 1: Treatments used are represented by [⁴⁴AANA⁴⁷]-CCL5 at 0.05–0.5–1 mg/kg ip. [⁴⁴AANA⁴⁷]- CCL5 at 1 mg/kg significantly down-modulated IFN-γ and TNF-a (*p<0.05). A trend to reduction for IL-6 and MCP-1. Panel 2: Treatments used are represented by Met- CCL5 at 0.05–0.5–1 mg/kg ip. Met- CCL5 at 1 mg/kg significantly reduced IL-6 (*p<0.05). Trend to reduction was observed for IFNγ and MCP-1. Results are given as mean ± SEM of n = 4 mice per group. All statistical analyses were performed using one-way ANOVA followed by Dunnett's multiple comparison post test (*p<0.05).

Figure 5. Evaluation of infiltrating inflammatory cells in ear sections.

Haematoxylin and Eosin staining on paraffin ear sections of Balb/c females 8–12 weeks of age sensitized and challenged in a DNFB-induced CHS. 30′ after challenge, mice were treated with [⁴⁴AANA⁴⁷]-CCL5 and Met-CCL5 at 0.05–0.5–1 mg/kg ip. Dexamethasone 0.5 mg/kg sc was used as reference compound. 4a) Vehicle group: high level of infiltration, polymorphonuclear cells (PMN) mostly, dendritic cells (DCs) and Natural Killer cells (NK) as lowest populations. A detail of hyperplasia of epithelium, represented by an increase in keratinocyte layer thickness is also shown. 4b) Cellular infiltrates and hyperplasia of epithelium are observed in ear sections of animals treated with [⁴⁴AANA⁴⁷]-CCL5 and Met-CCL5 at 0.05 mg/kg. 4c) [⁴⁴AANA⁴⁷]-CCL5 and Met-CCL5 at 0.5 mg/kg were able to decrease cellular infiltrates, hyperplasia of epithelium (detail shown in the panel) and dermis size (edema) to similar level as Dexamethasone. 4d) Reduction of cellular infiltrates and keratinocyte layers thickness is observed for [⁴⁴AANA⁴⁷]-CCL5 and Met-CCL5 at 1 mg/kg. 4e) Dexamethasone 0.5 mg/kg was used as reference compound. 4.1) Control group: not-sensitized, normal skin conditions. In all panels, magnification 20× ( scale bar: 16.5 µm), details 40× (scale bar: 20 µm).

Figure 6. Evaluation of CD3 infiltrates in ear sections.

Immunostaining with CD3 antibody on ear (paraffin sections) of Balb/c females 8–12 weeks of age sensitized and challenged in a Oxazolone-induced CHS. In all panels, magnification 40× (scale bar: 20 µm). A) Vehicle group: the CD3 staining on ear section revealed an important T cells involvement. B) No reduction of T cell infiltrates in ear sections is observed for [⁴⁴AANA⁴⁷]-CCL5 and Met-CCL5 at 0.05 mg/kg. C–D) [⁴⁴AANA⁴⁷]-CCL5 and Met-CCL5 at 0.5 and 1 mg/kg respectively were able to reduce T cell involvement to similar level as Dexamethasone. E) Dexamethasone 10 mg/kg was used as reference compound: clearly no staining is observed. F) Isotype control.

Figure 7. Expression of keratinocyte proliferation and differentiation markers in ear sections.

Immunostaining for keratins was performed on paraffin sections of ear skin of Balb/c females 8–12 weeks of age sensitized and challenged by Oxazolone. Treatments with [⁴⁴AANA⁴⁷]-CCL5 and Met-CCL5 0.5 mg/kg via i.p. took place 30′ post challenge. Dexamethasone 10 mg/kg sc was used as reference compound. A) Vehicle: important hyperproliferation of keratinocyte layer: K6 is widely expressed and it is found supra-basal, K14 is constitutively expressed. K10 showed an inverse staining compared to K6. B–C) [⁴⁴AANA⁴⁷]-CCL5 and Met-CCL5 0.5 mg/kg respectively were able to down-modulate K6 and K10 expression and to reduce hyperproliferation in a similar manner. K14 staining showed no changes. D) Dexamethasone 10 mg/kg used as reference compound: clearly no staining is observed. E) Isotype control. In all panels, magnification 20× (scale bar: 16.5 µm).