2010; Garland et al. airway epithelial cells. Our data underscore and designate the role of mouse CLCA1 in inflammatory airway disease to activate airway macrophages. In addition to its ability to upregulate cytokine expression which explains previous observations in the pneumonia mouse model, modulation of BPIFA1 expression expands the role of CLCA1 in airway disease to involvement in more complex downstream pathways, possibly including liquid homeostasis, airway protection, and antimicrobial defense. Electronic supplementary material The online version of this article (10.1007/s00418-018-1664-y) contains supplementary material, which is available to authorized users. possesses one functional gene copy in several mammalian species but two distinct functional copies in cattle, whereas in humans and in pigs it is a non-functional pseudogene Tlr4 (Gruber and Pauli 1999; Mundhenk et al. 2018; Plog et al. 2009). Interestingly, in the mouse, two gene duplication events resulted in three apparently functional CLCA3 proteins which are expressed in different cellular niches (Mundhenk et al. 2018; Patel et al. 2-Aminoheptane 2009). The gene is usually duplicated in the pig and the mouse with two or three, respectively, apparently distinctly regulated proteins, expressed in different cell types and functional niches (Patel et al. 2009; Plog et al. 2015). Other mammals appear to possess only a single CLCA4 protein (Plog et al. 2015). While such interspecies variations seem to be absent from CLCA1 around the genomic level, several functional differences have been described between human and mouse CLCA1 proteins. The human CLCA1 has been established as a key regulator of mucus cell metaplasia in inflammatory airway disease via interleukin (IL)-13-driven mucus gene transcription (Alevy et al. 2012). By contrast, a related mouse model failed to mirror the human data with regard to IL-13-dependence of CLCA1-mediated airway mucus production (Alevy et al. 2012). Moreover, CLCA1 modulates activation of a transmembrane protein 16A (TMEM16A, anoctamin-1)-mediated calcium-dependent chloride current (CaCC) in a paracrine and self-cleavage-dependent fashion (Sala-Rabanal et al. 2015). This non-cystic fibrosis transmembrane conductance regulator (CFTR)-mediated, option chloride current has been discussed as an important modulator of disease and therapeutic target in CF patients (Berschneider et al. 1988; Bronsveld et al. 2000; Taylor et al. 1988; Willumsen and Boucher 1989). However, tracheal instillation of IL-13 in murine airways resulted in overexpression of CLCA1 but failed to induce CaCC activity (Mundhenk et al. 2012). Moreover, CaCC was unchanged in the airways of 2-Aminoheptane deficiency resulted in decreased and responses with decreased neutrophil recruitment and reduced expression of the pro-inflammatory cytokine in a mouse model of acute (pneumonia (Dietert et al. 2014). By contrast, increased neutrophil recruitment preceded by CXCL-1 upregulation was seen following intranasal lipopolysaccharide (LPS) challenge in for 5?min at 4?C to remove remaining cells. Macromolecules were concentrated by centrifuging 10?ml of the collected media at 3000and 4?C through Vivaspin 15R columns of 5000 MWCO (Sartorius Stedim Biotech GmbH, Goettingen, Germany) to 100?l of conditioned medium (CM). All cell culture, transfection, supernatant collection, and Vivaspin concentration actions of CLCA1- and pcDNA-mock-transfected cell culture supernatants were performed identically. Hence, the pcDNA-CM contained the same amount of any secreted protein other than CLCA1. For macrophage stimulation, 50 or 100?l of CLCA1-CM or 100?l of pcDNA-CM as negative control was applied. Total protein concentrations were decided with the Micro BCA? Protein Assay Kit (Thermo Scientific, Rockford, IL, USA). CLCA1 was immunoprecipitated using the CLCA1-specific antibody -mCLCA3-C-1p (Bothe et al. 2011) followed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Specificity of this antibody was decided via Western Blot using -p3b2 antibody as described earlier 2-Aminoheptane (Bothe et al. 2011), shown in Online Resource 1a (Electronic Supplementary Material). 2-Aminoheptane The precipitated CLCA1 protein was also visualized on a Coomassie-stained SDS-PAGE gel compared to the pcDNA control, representatively shown in Online Resource 1b (Electronic Supplementary Material). Protein sizes were estimated using the Spectra? Multicolor Broad Range Protein Ladder (Thermo Scientific, Darmstadt, Germany). For a graphical illustration of the experimental setup, see Online Resource 2 (Electronic Supplementary Material). Isolation of murine alveolar macrophages WT and for 5?min at 4?C. The resulting pellet was resuspended in 400?l, complete medium, i.e., VLE RPMI 1640 Medium (VLE RPMI; Biochrom AG, Berlin, Germany), supplemented with 1% penicillinCstreptomycin (Biowest, Nuaill, France) and 10% FBS. 5??105 cells were seeded into each well of a 24-well plate. Medium was replaced after 2?h and cells were incubated for 24?h prior to stimulation (Kostadinova et al. 2016). To test the purity of the alveolar macrophage cell cultures, cells of the BAL were.