Broad Th2 neutralization and anti-inflammatory action of pentosan polysulfate sodium in experimental allergic rhinitis

Abstract

Background: Th2 cytokines like interleukin-4, -5, and -13 are regarded as important drivers of the immunopathology underlying allergic rhinitis (AR) and asthma. The present study explores the capacity of pentosan polysulfate sodium (PPS), a semi-synthetic heparin-like macromolecular carbohydrate, to bind Th2 cytokines and exert biological neutralization in vitro, as well as anti-inflammatory actions in vivo.

Methodology: The capacity of PPS to bind recombinant Th2 cytokines was tested with surface plasmon resonance (SPR) technology and biological Th2 neutralization was assessed by Th2-dependent proliferation assays. The in vivo anti-inflammatory action of PPS was studied using a validated Guinea-pig model of AR.

Results: Binding studies revealed a strong and specific binding of PPS to IL-4, IL-5, and IL-13 with IC values suggesting as stronger cytokine binding than for heparin. Cytokine binding translated to a biological neutralization as PPS dose dependently inhibited Th2-dependent cell proliferation. Topical administration of PPS 30 min prior to nasal allergen challenge of sensitized animals significantly reduced late phase plasma extravasation, luminal influx of eosinophils, neutrophils, and total lavage leukocytes. Similar, albeit not statistically secured, effects were found for tissue leukocytes and mucus hyper-secretion. The anti-inflammatory effects of PPS compared favorably with established topical nasal steroid treatment.

Conclusion: This study points out PPS as a potent Th2 cytokine-binding molecule with biological neutralization capacity and broad anti-inflammatory effects in vivo. As such PPS fulfills the role as a potential candidate molecule for the treatment of AR and further studies of clinical efficacy seems highly warranted.

Keywords: Allergy; Th2 cytokines; interleukin-13; interleukin-4; interleukin-5; pentosan polyphosphate sodium.

Figure 1

Schematic overview of the in vivo study design. Guinea pigs were sensitized to OVA at day 0 (sensitization 1) and day 7 (sensitization 2). Three weeks after the last sensitization, at day 28, animal groups were pre‐treated with either Budesonide (n = 10) or PPS (0.5 mg/kg, n = 10 or 5 mg/kg, n = 10) for 30 min prior to OVA or Saline challenge (control groups received PBS pre‐treatment, (n = 7 per control group). Changes in the late phase response were determined by analysis of nasal lavage fluid and histological nasal tissue (septum) at 8 h after allergen challenge.

Figure 2

Molecular interactions and binding of PPS and heparin to Th2 cytokines as revealed by surface plasmon resonance (SPR) methodology. Data are shown for representative titration experiments showing the capacity of soluble heparin (dashed lines) and PPS (solid lines) to bind (A) IL‐4, (B) IL‐5, and (C) IL‐13 and prevent attachment to a cytokine‐binding detector chip. The experiments were performed on the BIAcore 2000 platform and with a heparinised CM4 flow cell detector 29.

Figure 3

Dose–response curves of the capacity of PPS (solid line) and heparin (dashed line) to inhibit cytokine‐dependent cell proliferation in vitro. Data are shown for representative experiments. The cell densities were measured as luciferase activity 28 by a Victor 1420 Multi‐label counter (Wallac, Turku, Finland) after 48 h incubation in RPMI/5% 20 w/v FCS in the presence of 2.5 ng/mL stimulating cytokine: recombinant (A) IL‐4, (B) IL‐5, and (C) IL‐13.

Figure 4

Capacity of PPS and budesonide to reduce allergen‐induced influx of nasal lumen leukocytes (A–C) and plasma extravasation (D). Samples for leukocytes and plasma exudation were collected during the allergic late phase (8 h). Data are expressed as cells/mL nasal lavage fluid. Plasma extravasation was expressed as total protein levels in nasal fluid supernatants 30. Asterisks in brackets denote statistical difference between saline and OVA exposed positive controls whereas asterisks denote difference between sham versus drug‐treated OVA‐exposed animals. PPS‐0.5 and 5 correspond to a prophylactic topical dose of 0.5 and 5 mg/kg, respectively.

Figure 5

Impact of PPS and budesonide on histological nasal tissue parameters during the allergic late phase reaction. (A) Intraepithelial eosinophils (A), expressed as percent eosinophils per analysed tissue area and calculated by computerized image analysis of fluorochrome‐stained eosinophils (exemplified in E). Corresponding data for epithelial nasal tissue T lymphocytes are shown in B and G. Epithelial loss of PAS % mucin content is shown in C and H. Panel D shows a Pearson correlation analysis between epithelial T lymphocytes and epithelial loss of PAS positivity (i.e., mucus degranulation). (F) Hematoxylin stained nasal mucosa to exemplify the overall increased leukocyte tissue infiltration in allergen‐challenged animals (infiltrating leukocytes are exemplified by arrows). PPS‐0.5 and 5 correspond to a prophylactic topical dose of 0.5 and 5 mg/kg, respectively. OVA, ovalbumin; Sal, saline control. Bar codes in E–G = 70 µm, H = 50 µm.