This study documents that 4 week sub-chronic exposure to second hand cigarette smoke does not lead to measureable leukocyte infiltration within lung tissue but does result in airspace inflammation and decreased lung elastance. The inability of sub-chronic smoke exposure to promote lung tissue inflammation or changes in airspace structure is consistent with previous reports (D'Hulst et al., 2005
; Rinaldi et al., 2012
) and was observed in both C57BL/6 and Arhgef1−/−
animals. In contrast to the lack of change in lung tissue inflammation and architecture, SHS exposure elicits a robust increase in all leukocyte populations recovered from BAL as previously reported (Woodruff et al., 2009
). Within the SHS exposure cohort, all leukocyte populations examined were increased in Arhgef1−/−
airspace compared to C57BL/6. Characterization of lymphocyte subsets recovered from the BAL of SHS exposed animals also reveal a statistically significant increase in the Arhgef1−/−
samples compared to identically treated C57BL/6 samples. Together these data demonstrate that even sub-chronic (4 week) exposure to SHS is sufficient to induce significant increases in the number of airspace leukocytes present in either healthy or mutant lungs.
Examination of lung structure in C57BL/6 SHS exposed animals indicates that no morphological changes in lung structure have occurred. This result was not unexpected as it has been previously reported for this mouse strain that at least 3 months of tobacco smoke exposure are required to induce structural changes as measured by an increase in mean linear intercept (Bartalesi et al., 2005
). We did hypothesize that the presence of pre-existing inflammation and airspace enlargement in the naïve Arhgef1-deficient mice would decrease the duration of SHS exposure required to induce further pathological changes. However, our current 4 week protocol failed to induce any increase in airspace enlargement in the Arhgef1-deficient animal beyond what is already present in the naïve animals, despite the exaggerated inflammatory response of the Arhgef1−/−
mice to SHS exposure.
Significant decreases in lung elastance are evident in the C57BL/6 and Arhgef1−/−
animals when exposed to SHS for a relative short duration (4 weeks). Initially we were surprised to observe a change in lung mechanics with a relative short smoke exposure protocol. A previous report failed to observe any changes in the lung mechanics of C57BL/6 mice exposed to smoke for 6 months (Guerassimov et al., 2004
). However close examination of the methods employed to measure lung mechanics reveal an importance difference that may account, at least in part, for the discrepancy between our results and their study. In the previous report the investigators performed a primewave perturbation across a range of positive end expiratory pressures (PEEP) from 3 to 9 cm H2
O in order to generate a P-V loop (Guerassimov et al., 2004
). In our study we generated a pressure-volume loop where lung mechanics are measured over a range of pressures from 2 ~ 30 cm H2
O. Examination of our own pressure and volume measurements between 2 and 10 cm H2
O portion of the loop reveal no discernible difference in the SHS exposure groups. We did perform the primewave perturbation at a PEEP of 3 cm H2
O and consistent with their results do not detect any significant changes in lung elastance in the SHS exposed animals (data not shown). A recent review included both of these measurements performed in the pallid mouse strains (see Figure 4 in Wright et al., 2008
). The primewave perturbation yields a modest but significant shift in lung mechanics of the pallid strain (Figure 4A in Wright et al., 2008
) while the pressure-volume loop inflating the lungs up to pressures around 30 cm H2
O demonstrate dramatic differences between the healthy and diseased (pallid) lungs (Figure 4B in Wright et al., 2008
). Together these data suggest that a P-V loop which inflates the lungs up to higher pressures (~30 cm H2
O) may be more sensitive to detecting modest changes in lung elastance than measurement performed at lower pressures.
In addition to the differences in methods of measurements it is also worth noting the difference in duration of smoke exposure protocols between the studies, 4 weeks versus 6 months (Guerassimov et al., 2004
). Aside from the increased duration of smoke exposure another parameter to consider is the age of the mice at the time of assessment. Our studies performing lung mechanics measurements find a progressive decrease in lung elastance of C57BL/6 mice from 3 months of age to 1 year of age, similar to reports by other investigators (Huang et al., 2007
). Comparison of naïve Arhgef1−/−
mice and C57BL/6 mice across these ages reveal the most pronounced differences between strains occur at 6 months of age (Hartney et al., 2010
Comparing the lung mechanics and airspace architecture between all four groups suggests that changes in murine lung mechanics can occur in the presence or absence of changes in lung architecture. Note the SHS exposed C57BL/6 mice have lung elastance values lower than the naïve Arhgef1-deficient mice despite the lack of alteration in airspace structure, as measured by mean linear intercept (Figures and ). The lack of a direct correlation between lung mechanics and airspace structure has been noted by several investigators examining the effects of cigarette smoke exposure in mouse models (Guerassimov et al., 2004
; Foronjy et al., 2005
; Rinaldi et al., 2012
). Based on these differences it has been proposed that separate pathways are involved in the development of histological alterations in lung architecture versus physiological changes in lung mechanics (Foronjy et al., 2005
). Our study provides another instance of these two pulmonary phenotypes occurring independently and supports their proposed hypothesis.
We have previously described a signaling pathway that operates in pulmonary myeloid cells that leads to the production of pro-inflammatory mediators and is normally inhibited by Arhgef1 (Hartney et al., 2011
). To address whether this same Arhgef1-regulated pathway contributes to cigarette smoke-induced inflammation we compared the responses of Arhgef1−/−
and wild type mice to SHS exposure. Using a Two-Way ANOVA, no interaction is found between genotype and response to cigarette smoke exposure in any of our data sets. Thus, we conclude that these pathways, Arhgef1 and cigarette smoke exposure induced responses appear to occur independently of each other.
In conclusion the data presented here demonstrate that sub-chronic SHS exposure is sufficient to induce a significant increase in airspace leukocytes and decrease in lung elastance in both healthy animals and a mutant mouse strain with pre-existing pulmonary inflammation and pathology. This change in lung mechanics appears to occur as a result of processes that can be independent of changes in airspace structure. Further examination of the pathways responsible for SHS induced changes in lung mechanics may identify novel targets for restoring or retaining lung elastance in human subjects exposed to second hand smoke.
Conflict of interest statement
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.