The Absence of Mrp4 Has No Effect on the Recruitment of Neutrophils and Eosinophils into the Lung after LPS, Cigarette Smoke or Allergen Challenge

Abstract

The multidrug resistance protein 4 (Mrp4) is an ATP-binding cassette transporter that is capable of exporting the second messenger cAMP from cells, a process that might regulate cAMP-mediated anti-inflammatory processes. However, using LPS- or cigarette smoke (CS)-inflammation models, we found that neutrophil numbers in the bronchoalveolar lavage fluid (BALF) were similar in Mrp4−/− and Mrp4+/+ mice treated with LPS or CS. Similarly, neutrophil numbers were not reduced in the BALF of LPS-challenged wt mice after treatment with 10 or 30 mg/kg of the Mrp1/4 inhibitor MK571. The absence of Mrp4 also had no impact on the influx of eosinophils or IL-4 and IL-5 levels in the BALF after OVA airway challenge in mice sensitized with OVA/alum. LPS-induced cytokine release in whole blood ex vivo was also not affected by the absence of Mrp4. These data clearly suggest that Mrp4 deficiency alone is not sufficient to reduce inflammatory processes in vivo. We hypothesized that in combination with PDE4 inhibitors, used at suboptimal concentrations, the anti-inflammatory effect would be more pronounced. However, LPS-induced neutrophil recruitment into the lung was no different between Mrp4−/− and Mrp4+/+ mice treated with 3 mg/kg Roflumilast. Finally, the single and combined administration of 10 and 30 mg/kg MK571 and the specific breast cancer resistance protein (BCRP) inhibitor KO143 showed no reduction of LPS-induced TNFα release into the BALF compared to vehicle treated control animals. Similarly, LPS-induced TNFα release in murine whole blood of Mrp4+/+ or Mrp4−/− mice was not reduced by KO143 (1, 10 µM). Thus, BCRP seems not to be able to compensate for the absence or inhibition of Mrp4 in the used models. Taken together, our data suggest that Mrp4 is not essential for the recruitment of neutrophils into the lung after LPS or CS exposure or of eosinophils after allergen exposure.

Effects of conventional tobacco smoke and nicotine‐free cigarette smoke on airway inflammation, airway remodelling and lung function in a triple allergen model of severe asthma

“…Cigarette smoke exposure Mice were exposed to CS in a heated (38°C) whole body exposure chamber for either 6 weeks (5 days per week) in the chronic asthma model, or 4 days in the helminthic model as described previously [29]. Mice were exposed to 4 cigarettes (Roth-H€ andle without filters, tar 10 mg, nicotine 1 mg, carbon monoxide 6 mg, Imperial Tobacco) per day in the chronic asthma model, or six cigarettes per day in the helminthic infection model.…”

“…This triple allergen model, in which mice are immunized with a cocktail of ovalbumin (OVA), extracts of cockroach (CRA) and house dust mite (HDM) followed by twelve challenges (2 per week), alternating between allergens, developed a stronger Th2 type inflammation in the lung, enhanced loss of lung function, higher IgE serum concentrations and collagen I formation in the lung in comparison to only using OVA, CRA or HDM alone in single allergen exposure/challenge models [28]. In the current study, we combined this model with passive tCS exposure (as described previously [29]) by exposing sensitized mice to tCS five times a week during the 6 week allergen challenge period. In the current study, we combined this model with passive tCS exposure (as described previously [29]) by exposing sensitized mice to tCS five times a week during the 6 week allergen challenge period.…”

“…Furthermore, when mice immunized with all three allergens were challenged with a mixture of all three allergens on the six consecutive weeks following the immunization period, the resulting asthmatic phenotype was much weaker compared to mice where the allergens were alternated during the airway challenges. In the current study, we combined this model with passive tCS exposure (as described previously [29]) by exposing sensitized mice to tCS five times a week during the 6 week allergen challenge period. Our aim was to develop a model mimicking the pathology of asthma worsening observed in smoking asthmatics, including a strong neutrophilia plus eosinophilia, increased AHR, increased remodelling (collagen I formation, mucus production and smooth muscle thickening) and reduced sensitivity to steroid treatment compared to allergenonly exposed mice.…”