The effects of the anti-leukotriene zileuton upon neutrophil recruitment in X-linked chronic granulomatous disease knock-out mice exposed to sterile Aspergillus fumigatus hyphae

Chronic Granulomatous Disease (CGD) is a recessive disease caused by defects in the neutrophil respiratory burst oxidase that produces superoxide, an important precursor for microbicidal oxidants. CGD patients suffer from increased microbial and fungal infections as well as inflammatory granulomas that may represent persistent lesions in response to debris normally cleared by oxidant-dependent mechanisms, such as Aspergillus fumigatus hyphae (Morgenstern et. al., J Exp. Med 185, 207, 1997). An experimental model for CGD is a knock-out mouse with a mutation in the X-linked gene for gp91^pbox that codes for the larger subunit of the respiratory burst oxidase cytochrome b. We have previously shown that the inflammatory exudate induced by intraperitoneal (I.P.) injection of thioglycollate in X-CGD mice had increased numbers of neutrophils compared to wild-type (WT). Experimental evidence suggests oxidants are important for degradation of leukotrienes, including leukotriene B₄ (LTB₄), a potent neutrophil chemoattractant. We hypothesized that increased numbers of X-CGD neutrophils may be recruited by an accumulation of chemoattractants in the absence of their superoxide-dependent clearance or inactivation. In the current study Zileuton, a 5-Lipoxygenase inhibitor, was used to inhibit the production of LTB₄ via the arachidonic acid pathway. We investigated the effect of Zileuton on the inflammatory exudate induced by an I.P. injection of sterilized A. fumigatus hyphae (50 μg) in WT and X-CGD C57BL/6J mice. 30 minutes before injection and 3 hours after, either Zileuton (100 mg/kg suspended in 0.2% hydroxypropyl methylcellulose. HPMC) or 0.2% HPMC alone was administered by gavage. After 6 hours, when neutrophils were present in maximal numbers, mice were sacrificed and peritoneal cavities lavaged to assess exudate formation with total cell counts and histologic analysis of cellular composition. In mice administered the HPMC vehicle alone, the X-CGD mice (n=9, 6.03×10⁶ +/- 3.56×10⁶) compared to WT (n=13, 6.15×10⁵ +/- 6.42×10⁵) had a 10-fold increase in neutrophil numbers (p<0.0001). Zileuton decreased the numbers of exudate neutrophils isolated from WT (n=14, 2.56×10⁵ +/- 1.23×10⁵) and X-CGD mice (n=8, 1.40×10⁶ +/-6.42×10⁵), however neutrophils were still increased by 5-fold in X-CGD relative to WT mice (p<0.0001). These results suggest that leukotriene-elicited neutrophil chemotaxis of X-CGD in response to sterile A. fumigatus is an important mechanism for recruiting increased numbers of neutrophils in murine X-CGD, although other mechanisms may also contribute.