Acute chlorine gas exposure produces transient inflammation and a progressive alteration in surfactant composition with accompanying mechanical dysfunction

Abstract

Acute Cl2 exposure following industrial accidents or military/terrorist activity causes pulmonary injury and severe acute respiratory distress. Prior studies suggest that antioxidant depletion is important in producing dysfunction, however a pathophysiologic mechanism has not been elucidated. We propose that acute Cl2 inhalation leads to oxidative modification of lung lining fluid, producing surfactant inactivation, inflammation and mechanical respiratory dysfunction at the organ level. C57BL/6J mice underwent whole-body exposure to an effective 60ppm-hour Cl2 dose, and were euthanized 3, 24 and 48h later. Whereas pulmonary architecture and endothelial barrier function were preserved, transient neutrophilia, peaking at 24h, was noted. Increased expression of ARG1, CCL2, RETLNA, IL-1b, and PTGS2 genes was observed in bronchoalveolar lavage (BAL) cells with peak change in all genes at 24h. Cl2 exposure had no effect on NOS2 mRNA or iNOS protein expression, nor on BAL NO3(-) or NO2(-). Expression of the alternative macrophage activation markers, Relm-α and mannose receptor was increased in alveolar macrophages and pulmonary epithelium. Capillary surfactometry demonstrated impaired surfactant function, and altered BAL phospholipid and surfactant protein content following exposure. Organ level respiratory function was assessed by forced oscillation technique at 5 end expiratory pressures. Cl2 exposure had no significant effect on either airway or tissue resistance. Pulmonary elastance was elevated with time following exposure and demonstrated PEEP refractory derecruitment at 48h, despite waning inflammation. These data support a role for surfactant inactivation as a physiologic mechanism underlying respiratory dysfunction following Cl2 inhalation.

Keywords: Alternative activation; Bronchoalveolar lavage; Nitric oxide; Pulmonary mechanics; Respiratory impedance.

Figure 1

Measured Cl₂ concentration differs from target concentration and depends on the presence of mice within the system. In-chamber Cl₂ concentration was monitored in real time by drawing 0.6 L/min chamber gas into an ITX-4 multigas meter. Measurements were made either with no mice or 5 mice within the chamber. Line represents the logarithmic regression of the experimental data (R² = 0.9918 for no mice, 0.8607 for 5 mice) and is used to calculate the net exposure (ppm × time) by integrating the area under the curve (160 ppm-hr in the absence of mice and 60 ppm-hr in the presence).

Figure 2

Cl₂ exposure results in transient inflammation with preserved lung architecture. Inflation fixed lung tissue was paraffin embedded, sectioned and H+E stained. Whole lung field is shown using 8× magnification with 400× inset showing small airways and adjacent parenchyma. (A) 3 hours post exposure. (B) 24 hours post exposure. (C) 48 hours post exposure, (D) Air exposed. Note focal consolidation (arrow) at 24 hours, with inset (C) showing interstitial and airway luminal swelling and cellular infiltration. By 48 hours (D), infiltrate density was diminished and interstitial edema had waned, although airway luminal irregularity persisted.

Figure 3

Cl₂ exposure produces transient neutrophilia. Cytospin slides were stained with modified Wright-Giemsa stain. Cell differentials were obtained by examination of 5 non-contiguous high power fields per slide. Data are mean ± SE, n = 8 mice per condition. * denotes statistical significance versus Air Exposed (p<0.05 by Dunnet’s t-test following one-way ANOVA).

Figure 4

Cl₂ exposure increases Relm-α protein in the pulmonary epithelium. Differential expression and cellular localization of the alternative-activation marker Relm-α was determined by immunohistochemical assay following Cl₂ exposure. (A) 3 Hours, (B) 24 Hours, (C) 48 Hours, (D) Air Exposed.

Figure 5

Cl₂ exposure increases Mannose Receptor protein expression in the pulmonary epithelium and alveolar macrophage. Differential expression and cellular localization of the Mannose Receptor was determined by immunohistochemical assay following Cl₂ exposure. (A) 3 Hours, (B) 24 Hours, (C) 48 Hours, (D) Air Exposed.

Figure 6

Cl₂ exposure alters BAL surfactant content and function. Upper panel: BAL phospholipid content was determined as described. Middle panel: Surfactant function was assessed by determining initial opening pressure in a capillary surfactometer containing the large aggregate fraction of BAL using equal phospholipid loading across all samples. Lower panel: BAL content of SP-D and SP-B was assessed by western blot. Relative SP-D to SP-B content was estimated by densitometry of the band intensity, normalizing to air exposed, and calculating the SP-D to SP-B ratio. All data are presented as mean ± SE, n = 4. * denotes statistical significance versus Air Exposed (p<0.05) using t-test following 1-way ANOVA.

Figure 7

Cl₂ exposure produces PEEP dependent alteration to lung mechanical function. Lung Elastance (E_L – left column) and Resistance (R_L – right column) are presented between 0.5 and 20 Hz to allow independent partitioning of airway and parenchymal effects. Data points represent the mean ± SE of the experimental data, n = 8 mice per condition. Smooth lines represent the model fit to the data as described in the methods. Air exposed – filled circles and solid line; 3 hours post Cl₂ exposure – hollow circles and dotted line; 24 hours post Cl₂ exposure – filled square and dashed line; 48 hours post Cl₂ exposure – hollow square and dash and double-dotted line.

Figure 8

Cl₂ exposure produces PEEP dependent alteration to quasi-static pressure volume relationships. Left: Quasi-static pressure volume loops generated from a PEEP of 1 cm H₂O display increased PV-hysteresis at 24 hours following injury, a trend seen for all levels of PEEP. Curves represent the mean of 8 mice for each exposure condition; variance is not displayed for clarity. PV loops at other levels of PEEP are available in the online supplement. Right: PV loop areas were determined by integrating the volume with respect to pressure as a measure of hysteresis. PV area is significantly elevated at all levels of PEEP at 24 hours following exposure. Data are presented as mean ± SE.