ENO and ECO in naïve mice
Average levels of ENO in naïve C57Bl6 mice from 8–22 weeks of age ranged from 5–7 ppb (Figure ). ENO levels in naïve A/J, MKK3-/-, MKK3-sufficient (C57/C129) and NOS-3-/- mice were not statistically different from the C57BL6 mice, ranging from 6–9 ppb. However, ENO was significantly elevated for IL-10-/- mice, and unexpectedly, for NOS-2-/- mice, to levels of 8–10 and 15–17 ppb (P < 0.05), respectively. Notably, ENO levels were significantly decreased in JNK1-/- mice. These data suggested that non-invasively measured ENO was mouse strain and genetic-knockout dependent, prior to any experimental manipulations.
Figure 2 A. Exhaled NO (ENO) in naïve mice of various strains at 8–22 weeks of age. Values are means ± SE; *P < 0.05 vs. C57Bl6; vP < 0.05 JNK1-/- vs. MKK3-/-; ^P <0.05 NOS-2-/- vs. NOS-3-/-; strain(n) = C57Bl6(19), (more ...)
Average levels of ECO in naïve C57Bl6 mice ranged from 2–2.5 ppm (Figure ). ECO levels in naïve IL-10-/-, JNK1-/-, and NOS-3-/- mice were not statistically different from the C57Bl6 mice, ranging from 1.5–2.5 ppm. However, ECO was significantly elevated in the A/J (~3.0 ± 0.2 ppm, P < 0.05), MKK3-/- (~4.0 ± 0.2 ppm, P < 0.05), MKK3-sufficient (~3.2 ± 0.2 ppm, P < 0.05), and NOS-2-/- (~3.1 ± 0.1 ppm, P < 0.05) mice. Furthermore, ECO was significantly increased in the MKK3-/- mice as compared to wild-type littermate controls (~4.1 ± 0.2 vs. 3.2 ± 0.2 ppm, P < 0.05), and likewise was significantly increased in the NOS-2-/-, as compared to NOS-3-/-, mice (3.4 ± 0.1 vs. 2.8 ± 0.1 ppm, P < 0.05). Similar to ENO, these data suggested that non-invasively measured ECO was mouse strain and genetic-knockout dependent, prior to any experimental manipulations.
AR in naïve mice
AR to inhaled MCh was demonstrated in naïve C57Bl6 control mice, such that Penh was significantly increased at all concentrations ≥ 10 mg/ml (Figure ). Likewise, naïve IL-10-/- mice demonstrated significant AR, which was of decreased magnitude as compared to the IL-10-sufficient wild-type C57Bl6 mice. NOS-3-/- mice demonstrated a trend toward increased AR at these same MCh concentrations, which was not significantly different from respective 0 MCh, and was significantly lower than C57Bl6 controls at MCh concentrations above 10 mg/ml (P < 0.05). NOS-2-/- mice displayed a nearly flat AR relationship, which was likewise significantly lower (as indicated by inverted carat) than control C57Bl6 mice at MCh concentrations above 10 mg/ml (P < 0.05), and was significantly lower than the NOS-3-/- mice at MCh concentrations ≥ 10 mg/ml (P < 0.05).
Figure 3 Airway responses to MCh (as enhanced pause; Penh) in naïve mice of various strains. A. Means ± SE shown for strains C57Bl6 (filled circle, n = 12), IL-10-/- (open circle, n = 12), NOS-2-/- (filled triangle, n = 8) and NOS-3-/- (open triangle, (more ...)
Shown on a greater scale, with C57Bl6 mice for reference, naïve A/J mice demonstrated significant AR to MCh at all concentrations above 1 mg/ml, and this response was significantly greater than that observed in C57Bl6 mice, at these same concentrations (Figure ). Interestingly, MKK3-/- mice likewise displayed significant AR at MCh concentrations ≥ 10 mg/ml, and were found to be increased at the highest MCh concentration (50 mg/ml; P < 0.05) as compared to C57Bl6 controls. JNK1-/- mice also showed significant AR at 25 and 50 mg/ml, and were significantly less than C57Bl6 mice at the highest concentration of MCh (50 mg/ml, P < 0.05). The MKK3-sufficient littermates (C57/C129 mice) were relatively flat in MCh response, demonstrating a small but significant increase only at the highest MCh concentration. These non-invasively obtained AR are consistent with potential mechanisms dependent on airway ENO and ECO levels, indicated by our exhaled gas measurements.
ENO and ECO as a function of age
ENO was measured in naive IL-10-/- mice (n = 234) from ages 8 to 58 weeks (Figure ). The non-linear regression demonstrated a significant curvilinear relationship of ENO with age, beginning at a high point of 13.8 ppb (the y-intercept at 8 weeks of age) and declining to an asymptote of 5 ppb, by 58 weeks of age. Unlike the IL-10-/- mice, the naïve C57Bl6 mice tested (n = 51) had a lower y-intercept (7.7. ppb at 8 weeks of age) and demonstrated no significant change in ENO from ages 8–50 weeks of age, as indicated by a calculated slope value that was near zero over the age range studied (Figure ). These results suggest that non-invasively measured ENO is stable with age in naïve C57Bl6 mice, but is high and falls with age in the absence of IL-10 and experimental manipulations.
Figure 4 ENO and ECO as a function of age. A. ENO with age in IL-10-/- mice (n = 234) from 8–58 weeks of age. Line indicates best-fit exponential decay regression curve (P < 0.05), calculated as described in the Methods section. Calculated regression (more ...)
Unlike ENO in the IL-10-/- mice, ECO was relatively constant with age in the naïve IL-10-/- and C57Bl6 mice tested (Figures and ). The y-intercepts at 8 weeks were significantly different from 0 ppm ECO in each case (3.7 and 2.1, respectively), but the slopes of the regression lines were not significantly different from zero, suggesting no significant change over the aging interval monitored.
ENO and ECO with induction of allergic airway inflammation
Allergic airway inflammation, as reflected by BAL total cell count, and macrophage and eosinophil numbers, was produced to varying degrees in all mice with systemic sensitization and airway challenge using OVA (Table ); however, the total cell count and eosinophil responses were minimal in NOS-2-/- mice. With allergic airway inflammation, a group of C57Bl6 mice (n = 20) followed over time, from the naïve state, through systemic OVA sensitization injection, and subsequent OVA aerosol airway challenge, demonstrated a significant rise in non-invasively measured ENO over the course of the inflammation induction protocol. Figure indicates an average starting point of nearly 7 ppb, with a significant doubling to approximately 12 (P < 0.05) and 14 (P < 0.05) ppb, at 24 and 48 hr after the final OVA aerosol challenge, followed by a decline to approximately 10 ppb at 72 hr (n.s.). This pattern was suggestive of an early to intermediate response of ENO with induction of allergic airway inflammation, followed by a reduction in the late-phase portion of the inflammatory response.
Bronchoalveolar lavage cell counts in various strains of mice.
Figure 5 Alterations in ENO and ECO with induction of inflammation. A. Changes in ENO with induction of airway inflammation by OVA treatment in C57Bl6 mice, over duration of sensitization and challenge protocol (3 wk.). Naïve is prior to systemic OVA sensitization (more ...)
Similarly, in those same mice, ECO was increased with induction of allergic airway inflammation, rising from 2.3 ppm in the naïve state, to 2.8 ppm over the 24–72 hr time interval after the final OVA aerosol challenge (Figure ). This pattern was consistent with an early response of ECO with induction of allergic airway inflammation, but lacked the fall at 72 hr observed with ENO, suggesting a lack of resolution within the same time interval as that observed for ENO.
ENO and ECO comparisons with allergic airway inflammation
With allergic airway inflammation, the levels of ENO significantly increased from 1–3-fold (P < 0.05) in C57Bl6, A/J, MKK3-/-, MKK3-sufficient, and JNK1-/- mice (Figure ), when compared to ENO values from each respective naïve strain or knockout. The numerical increase in ENO in IL-10-/- mice suggested a similar increase, but was not statistically different from the naïve IL-10-/- ENO values. ENO of the MKK3-/- mice with allergic airway inflammation was greater (P < 0.05) than its MKK3-sufficient littermate control. However, as compared to both C57Bl6 and NOS-3-/- mice, the NOS-2-/- mice demonstrated a significant reduction in ENO with allergic airway inflammation (-50%, P < 0.05), while ENO of the NOS-3-/- mice remained unchanged from baseline levels and was different than the NOS-2-/- mice. These data suggested that different responses to allergic airway inflammation were dependent on the strain or targeted gene knockout of the mice tested, as measured with our non-invasive technique.
Figure 6 Comparisons of ENO and ECO across mouse strains. A. Exhaled NO (ENO) with airway inflammation, expressed as fold change as compared to ENO in respective-strain naïve mice. *P < 0.05 vs. respective-strain naïve mice controls; + (more ...)
ECO was significantly increased ~1–2.5-fold in C57Bl6, IL-10-/-, A/J, and JNK1-/- mice with airway inflammation (Figure ), as compared to respective naïve strain or knockout mice. No significant change in ECO was observed in MKK3-/-, MKK3-sufficient, NOS-2-/-, and NOS-3-/- mice, compared to their appropriate littermate controls. These data suggested different responses of ECO as compared to ENO, as a function of allergic airway inflammation and mouse strain or targeted gene knockout.
Effects of NOS and HO inhibition
As noted previously in Figure , systemic sensitization with i.p. injections of OVA in untreated C57Bl6 control mice did not significantly increase ECO, but challenge with OVA aerosol resulted in an increase in ECO, indicating that the rise in ECO was associated with induction of allergic airway inflammation (Figure ). This increase in ECO was attenuated in mice pretreated with SnPP to inhibit HO. The degree of attenuation made it statistically similar to both the non-SnPP-treated group with airway inflammation, and the non-SnPP-treated naïve controls, which suggested that the value was intermediate between them.
Figure 7 Changes in ECO and AR with inhibition of HO. A. Exhaled CO (ECO) changes with inhibition of heme-oxygenase by tin protoporphyrin (SnPP) without induction of airway inflammation (OVA, i.p.) and with airway inflammation (+OVA aerosol), in C57Bl6 mice, expressed (more ...)
As compared to naïve C57Bl6 mice, AR increased at the highest concentration of MCh (50 mg/ml, P < 0.05) in C57Bl6 mice with allergic airway inflammation due to OVA (Figure ). With SnPP administration in the presence of allergic airway inflammation, AR was also elevated at the highest concentration of MCh as compared to naïve controls (P < 0.05), but was not statistically different from that measured in mice treated with OVA and no SnPP. These data were suggestive of a trend toward increased AR with inhibition of HO in the presence of allergic airway inflammation.
In naive NOS-2-/- mice, a significant reduction in ENO from a median value of 22.5 ppb to 4 ppb (P < 0.05) was observed after chemical inhibition of neuronal/constitutive NOS (I) with SMTC (Figure ). These declines in ENO with SMTC administration were associated with a significant rise in ECO from a median value of 3.1 to 4.1 ppm (P < 0.05). Conversely, administration of SnPP to naïve NOS-2-/- mice resulted in a decline in ECO in each of the three mice tested, along with concomitant increases in ENO in those same mice (Figure ). Statistical analyses were not applied to those data due to the small number of mice tested, however, the respective ECO and ENO responses were similar in direction and magnitude. These data indicated that in the absence of NOS-2, inhibition of NO production by NOS-I can result in increases in CO levels, and that inhibition of CO production by HO may result in increased NO levels.
Figure 8 Modulation of ENO, ECO, and AR in NOS-2-/- mice. A. Changes in exhaled NO (ENO; open circle) and CO (ECO; filled circle) in NOS-2-/- mice (n = 4) 48 hr. after administration of S-methyl-L-thiocitrulline (SMTC). Open bar indicates median ENO value, black (more ...)
AR was increased in NOS-2-/- mice with induction of allergic airway inflammation by OVA (Figure ), which was consistent with the reduction of ENO observed in this group. AR was also increased in naive NOS-2-/- mice with inhibition of HO by SnPP. However, inhibition of NOS-I with SMTC did not alter AR to MCh as compared to naive NOS-2-/- mice, remaining flat across MCh concentrations. These data suggested that NO-inhibition driven changes in ECO may be associated with alterations in AR to MCh.