Differential disease states between distinct congenic mouse strains harboring identical mutations and maintained in a common environment provides a powerful means for identifying secondary genetic factors that have influence disease phenotypes. Here we report that the lungs of 20-day old congenic C57BL/6J CF mice, that progress to overt inflammatory disease, maintained in a sterile environment have elevated numbers of neutrophils and a corresponding increased level of both S100A8 and S100A9, which is not detected in other CF-affected tissues (ileum and liver). In contrast, the lungs of 20-day old congenic BALB/cJ CF mice, which do not develop any obvious inflammatory phenotype, housed with the congenic C57BL/6J CF animals, had no increase in S100A8 levels, although resident airway neutrophil numbers and S100A9 levels were similarly elevated.
S100A8 (calgranulin A, MRP8) and S100A9 (calgranulin B, MRP14) are small cytoplasmic proteins (members of the S100 family of the EF hand calcium-binding proteins ) that are expressed principally, constitutively and coordinately by circulating neutrophils and monocytes but not normally in tissue macrophages or lymphocytes . The two proteins make up roughly 30% of the cytosolic protein in these cells  and support distinct functions (both as monomers and homodimers), as well as forming calprotectin (S100A8/S100A9 heterodimer) in the presence of Ca2+, with potentially different function(s). Although an understanding of the complete role(s) of each of S100A8, S100A9 and calprotectin is currently lacking [57, 59] diverse functions that could impact on CF lung disease have been attributed to them, including calcium sensing , cell differentiation, arachidonic acid metabolism [61, 62], as well as leukocyte and monocyte endothelial microvascular adherence, transmigration and retention [63–69]. Moreover, calprotectin is implicated in bacteriostasis (reviewed in [44–46, 51, 70]), possibly by sequestering Zn2+ [71–78] as well as inhibiting the adhesion of bacteria to mucosal epithelial cells . S100A8's important role in regulating inflammatory processes is clearly indicated in S100A8-null mice, where loss of immunoprotection from invading maternal cells results in embryonic death shortly after implantation .
During chronic inflammatory conditions, including that underlying CF lung disease, S100A8 and S100A9 are coordinately upregulated and secreted into the extracellular milieu [57, 81], and their products elevated in the serum of patients [82–87], (reviewed in ). Likewise, coordinate regulation of S100A8 and S100A9 is also observed in neutrophils where absence of S100A9 leads to a coordinate loss of S100A8 expression [60, 88]. However, the concise mechanisms of regulatory controls that underlay S100A8 and S100A9 expression are unclear, although they are known to be complex and involve proinflammatory mediators including lipopolysaccharides , TNF, IFN-γ and IL-1 [90, 91].
The results of the present study are important to further understand the basis and pathogenesis of the inflammatory lung phenotype of CF mice, its distinction among different congenic strains and possibly having implications to understanding airway disease of CF patients. Several important points can be drawn from these results. First, these results provide further support for the increasingly prevalent notion of spontaneous inflammation of the CF airways. This conclusion is supported by: 1) the early incidence of elevations in S100A8 and S100A9 expression along with resident neutrophil influx, 2) the fact that the mice were maintained in sterile environments without detectable lung pathogens, and 3) elevated S100A8 levels were detected in the B6-CF lungs but not Bc-CF airways maintained in identical environments.
Second, since S100A8 and S100A9 act as potent leukocyte chemokines and their elevation at 20-days of age are the earliest reported signs of a lung inflammatory phenotype in CF mice, this elevation may be directly responsible for eliciting the massive neutrophil influx observed in 4–5 week old B6-CF lungs [26, 27, 42].
Third, these results implicate S100A8 alone or both S100A8/S100A9 (calprotectin), but not S100A9 alone, as having a possible role in progression of the inflammatory lung phenotype in CF mice.
Finally, since both the B6-CF and Bc-CF mice were maintained in identical environments, the differential levels of S100A8 expression between the two strains is likely influenced by secondary genetic factors acting on neutrophils (either intrinsically or through the pulmonary interstitial milieu) to either suppress or upregulate its expression in the Bc or B6 strain, respectively, rather than the effect of differential environmental exposures or infection status. However, since the elevated levels of S100A8 in the B6-CF lungs agrees with the corresponding increased population of neutrophils and no expression was detected in inducible cells (endothelial and macrophage), it is more likely that its expression is being suppressed in the Bc strain as opposed to B6-CF, which maintains expression in resident neutrophils. Since S100A8 is normally expressed in circulating but not interstitial neutrophils , a possible explanation for the differential S100A8 levels is that B6-CF neutrophils do not properly recognize or transition to the resident milieu of the CF lung, or their mechanism of suppression may be compromised; thereby, B6-CF neutrophils fail to properly down-regulate S100A8 expression once they leave circulation and enter the lung interstitium, which may constitute a basic defect of the neutrophils or lung in the absence of CFTR function. In this regard, further studies of differences between the B6-CF and Bc-CF lungs in terms of signaling pathways and the mechanisms underlying the neutrophil phenotype transition from circulatory to interstitial, as well as the effect of differential lung milieus on this transition will be required to ascertain the mechanistic basis of this defect.
The results of this study extend on two previous reports of S100A8 overexpression in the lungs of distinct CF mouse lines [31, 38]. In the first study by Thomas et al. , a constitutive 4-fold overexpression of S100A8 was detected in the lungs of CF mice homozygous for the G551D mutation (in which a spontaneous lung inflammatory phenotype has not been reported) compared to controls. Although expression of S100A9 was not investigated, the results suggested that CF pathology relates to abnormal regulation of the immune system. Importantly, however, this report documented significant variations in basal expression of S100A8 between individual G551D CF lungs, and since these mice were of a mixed 129/Sv × CD1 strain the differences was attributed to genetic variations. It is thus possible that the same genetic factor(s) conferring marked differences in S100A8 expression between congenic C57BL/6 and BALB/c CF lungs correspond to those of the former study, and that the consistent overexpression inherent to the congenic lines (as opposed to the variability of the mixed background) are necessary for the clear and consistent detection of a lung inflammatory phenotype. In the study by Xu et al. , a series of microarray analyses were performed to identify differential gene responses to the loss of CFTR in the lungs of FVB/N X C57BL/6 mixed background mice. Of the multiple genes identified as having significantly up- or down-regulated expression in the CF lungs, both S100A8 and S100A9 were found to be roughly 2-fold elevated. However, the specific cells conferring the overexpression and its possible effect on a lung inflammatory phenotype were not investigated. Moreover, since these studies were similarly performed on mixed background mice that would likely also have marked variability in S100A8 and/or S100A9 levels, the potential effect of the overexpression on the lung phenotype could not be readily assessed, in contrast to the strictly controlled aspects of the present investigation.
The results presented here justify additional studies to clarify the role of S100A8 overexpression on the pathogenesis and/or progression of the CF lung inflammatory disease, and, in particular, the possible effect of S100A8 inhibition.