Lipoxin A4 stable analogs reduce allergic airway responses via mechanisms distinct from CysLT1 receptor antagonism

Abstract

Cellular recruitment during inflammatory/immune responses is tightly regulated. The ability to dampen inflammation is imperative for prevention of chronic immune responses, as in asthma. Here we investigated the ability of lipoxin A4 (LXA4) stable analogs to regulate airway responses in two allergen-driven models of inflammation. A 15-epi-LXA4 analog (ATLa) and a 3-oxa-15-epi-LXA4 analog (ZK-994) prevented excessive eosinophil and T lymphocyte accumulation and activation after mice were sensitized and aerosol-challenged with ovalbumin. At <0.5 mg/kg, these LXA4 analogs reduced leukocyte trafficking into the lung by >50% and to a greater extent than equivalent doses of the CysLT1 receptor antagonist montelukast. Distinct from montelukast, ATLa treatment led to marked reductions in cysteinyl leukotrienes, interleukin-4 (IL-4), and IL-10, and both ATLa and ZK-994 inhibited levels of IL-13. In cockroach allergen-induced airway responses, both intraperitoneal and oral administration of ZK-994 significantly reduced parameters of airway inflammation and hyper-responsiveness in a dose-dependent manner. ZK-994 also significantly changed the balance of Th1/Th2-specific cytokine levels. Thus, the ATLa/LXA4 analog actions are distinct from CysLT1 antagonism and potently block both allergic airway inflammation and hyper-reactivity. Moreover, these results demonstrate these analogs' therapeutic potential as new agonists for the resolution of inflammation.

Figure 1

Structure of LXA₄ stable analogs. Structural analogs of LXA₄ were prepared to resist rapid conversion by PGDH and ω-oxidation as well as β-oxidation (ATLa) (see text for further details). In addition, the tetraene was converted to a trienyne to enhance chemical stability in light and acid (3-oxa-LXA₄).

Figure 2

LX stable analogs differ from montelukast in the regulation of allergen-induced airway inflammation. BAL fluids were obtained from OVA-sensitized and challenged mice that had been treated with ~500 mg/kg ATLa, ZK994, or montelukast by gavage. a) Eos and b) Lymphs were enumerated and identified after Wright-Giemsa stain. Results are expressed as means ± se (n≥6). c–e) Lipid mediators, cytokine and chemokine amounts in BAL fluids from animals treated with vehicle, montelukast, or LX analogs were determined by sensitive and specific EIAs. Results are expressed as the mean ± se (n≥6, duplicate determinations). *P < 0.05 by Student’s t test vs. vehicle control, ^‡P < 0.01 vs. montelukast, and ^†P < 0.03 vs. ATLa-treated animals.

Figure 3

Lung histopathology from montelukast and ATLa-treated mice. Male FVB mice were sensitized and aerosol-challenged with OVA in the absence (top row) or presence of ~500 µg/kg of either montelukast (middle row) or ATLa (bottom row). Representative (n=3) lung tissue sections were obtained from formalin-fixed, paraffin-embedded lung tissue prepared and stained with H&E. Magnifications, ×16 (left) and ×40 (right). Br, bronchus.

Figure 4

Oral ATLa stable analog reduces both airway hyper-responsiveness and inflammation. Mice were sensitized and challenged with CRA in the absence (white) or presence of ATLa [1 µg/ml (hatched) or 10 µg/ml (black)] in the drinking water. a) Airway reactivity was determined by methacholine-dependent change in lung resistance. Results are expressed as mean ± se (n≥5). b) Lungs were homogenized and levels of peptide mediators in the aqueous extracts were determined by sensitive and specific EIAs. Results are expressed as the mean ± se (n≥4, d≥2). *P < 0.05 by Student’s t test vs. vehicle control.

Figure 5

3-Oxa, trienyne-containing LXA₄ stable analog reduces airway inflammation induced by cockroach allergy. Mice were sensitized and challenged with CRA in the absence (white) or presence (black) of ZK-994 (100–1000 µg/kg, i.p.). a) Lung tissue sections were obtained from formalin-fixed, paraffin-embedded lung tissue prepared and stained with H&E, and b) peribronchial eosinophil accumulation was determined by morphometry (see Materials and Methods). Results are expressed as mean ± se (n≥6, d≥2), *P < 0.05 by Student’s t test vs. vehicle control.

Figure 6

ZK-994 regulates cytokine and chemokine mediators of Th2 adaptive immune responses to CRA. Mice were sensitized and challenged with CRA in the absence (white) or presence (black) of 3-oxa-LXA₄. a–c) Lungs from animals receiving ZK-994 (1000 µg/kg, i.p.) or vehicle control were homogenized and levels of peptide mediators in the aqueous extracts were determined by sensitive and specific EIAs. Results are expressed as the mean ± se (n≥6, d≥2), *P < 0.05 by Student’s t test vs. vehicle control.

Figure 7

Systemic and oral administration of ZK-994 reduces airway hyper-responsiveness. Cockroach-sensitized mice were treated by a) i.p. injection of vehicle (white) or ZK-994 (100 (gray), 400 (hatched), or 1000 (black) µg/kg, i.p.) or b) 10 µg/ml of either ZK-994 (black) or montelukast (gray), in a blinded manner, in the drinking water before allergen challenge. Airway reactivity was determined by a methacholine-dependent change in lung resistance. Results are expressed as mean ± se (n>5). *P < 0.05 by Student’s t test compared with control animals.