Cytochrome b5 and cytokeratin 17 are biomarkers in bronchoalveolar fluid signifying onset of acute lung injury

Abstract

Acute lung injury (ALI) is characterized by pulmonary edema and acute inflammation leading to pulmonary dysfunction and potentially death. Early medical intervention may ameliorate the severity of ALI, but unfortunately, there are no reliable biomarkers for early diagnosis. We screened for biomarkers in a mouse model of ALI. In this model, inhalation of S. aureus enterotoxin A causes increased capillary permeability, cell damage, and increase protein and cytokine concentration in the lungs. We set out to find predictive biomarkers of ALI in bronchoalveolar lavage (BAL) fluid before the onset of clinical manifestations. A cutting edge proteomic approach was used to compare BAL fluid harvested 16 h post S. aureus enterotoxin A inhalation versus BAL fluid from vehicle alone treated mice. The proteomic PF 2D platform permitted comparative analysis of proteomic maps and mass spectrometry identified cytochrome b5 and cytokeratin 17 in BAL fluid of mice challenged with S. aureus enterotoxin A. Validation of cytochrome b5 showed tropic expression in epithelial cells of the bronchioles. Importantly, S. aureus enterotoxin A inhalation significantly decreased cytochrome b5 during the onset of lung injury. Validation of cytokeratin 17 showed ubiquitous expression in lung tissue and increased presence in BAL fluid after S. aureus enterotoxin A inhalation. Therefore, these new biomarkers may be predictive of ALI onset in patients and could provide insight regarding the basis of lung injury and inflammation.

Figure 1. Intranasal SEA challenge induces lung injury and leukocyte infiltrate.

Mice received 1 µg of SEA diluted in BSS (Vehicle) through the i.n. route. (A) Lungs from day 2 SEA challenged mice were isolated, sectioned, and stained with H&E. Left panels display a low magnification (100X) and right panels high magnification (400X) of representative lung sections. Black arrows indicate leakage of red blood cells, white arrows indicate eosin staining refractile material (protein). (B) High magnification (400X) of lungs with (left panel) and without perfusion (right panel) of representative lung sections after vehicle alone and SEA i.n inhalation. Arrows indicate infiltrating leukocytes retained in blood vessel after perfusion. (C) Electron microscopy of lung blood vessels after vehicle alone and SEA i.n inhalation: Alveoli (Alv), Blood Vessel (B.Ves.), Erythrocyte (e), Leukocyte (L), Transmigrating leukocyte (L*). Bar  =  10 µm. The data are representative of three independent experiments using 2–4 mice per group.

Figure 2. Intranasal SEA challenge induces cellular infiltration, protein leakage and inflammation.

Mice received 1 µg of SEA or vehicle alone i.n., secondary (2°) challenge of either vehicle or SEA 48 h after the primary (1°) and were sacrificed 5 h later (53 hrs total). (A) Cells present in BAL fluid were harvested and enumerated and (B) total BAL fluid protein quantified by BCA. (C) IFNγ in BAL fluid and (D) in serum, and (E) IL-6 in BAL and (F) in serum were quantified by ELISA. (G) LDH in BAL fluid was quantified. The data are representative of two independent experiments using 4 mice per group. The errors bars indicate the standard error of the mean between biological replicates.

Figure 3. Differential proteomic fingerprint of BAL fluid from SEA vs. vehicle alone injected mice.

Mice were immunized as described in the legend of figure 1. After 16 h BAL fluid was obtained and 2 mg of protein per sample was processed on the Beckman Coulter ProteomeLab PF 2D platform. Chromatofocusing was performed as a linear gradient from pH 8.0 to pH 4.0. Fractions were collected in 0.3 pH intervals, automatically reinjected for a second dimension on a C18 column at 50°C. (A) Two-dimensional proteomic maps of BAL fluid from SEA and vehicle alone immunized mice, representative of one out three experiments is shown. (B) Chromatograms of second dimension fractions from SEA treated mice were overlaid with their corresponding equivalents from vehicle alone-injected mice. Overlays revealed peaks present in two fractions of the SEA treated samples but undetectable in the vehicle alone injected samples. (C) The fractions were lyophilized and resolved by 4–15% SDS-PAGE. Bands detected by a protein-specific fluorescent dye were cut out, digested by trypsin, and identified by LC/MS/MS. (D) Peptides sequences were searched against the NCBInr database version 20060804 using the Proteometrics Software Suite and the Profound Search Algorithm. The data are representative of three independent biological replicates. Each sample run on the PF 2D was a pool of BAL fluid obtained from 5 mice.

Figure 4. Cytochrome b5 expression is restricted to bronchiole epithelium in lungs.

Mice were immunized i.n. with 1 µg of SEA or vehicle alone i.n., secondary (2°) challenge of either vehicle or SEA 48 h after the primary, and were sacrificed 5 h later (53 hrs total). (A) Immunostaining of the lungs was performed using a rabbit polyclonal antibody against cytochrome b5 (Cyt b5) and rabbit immunoglobulin control (Ig). The data are representative of three independent experiments with 2–4 mice per group, magnification 100X. (B) The percentage of positive cytochrome b5 staining per bronchiole is reported for each sample by analyzing 30 representative bronchioles from two separate experiments. The errors bars indicate the standard error of the mean. **p<0.001.

Figure 5. Detection of cytokeratin 17 in BAL fluid after i.n. SEA.

Mice were immunized i.n. as described in legend of figure 4. (A) Immunostaining of the lungs was performed using a rabbit polyclonal antibody against cytokeratin 17 (CK17) and rabbit immunoglobulin control (Ig). The data are representative of three independent experiments, magnification 100X. (B) Immunoblotting of 5x concentrated BAL fluid was performed using a rabbit polyclonal against cytokeratin 17 (CK17) and rabbit IgG control. (C) BAL fluid harvested at different times after i.n. SEA immunization and BAL fluid from naïve mice were immunoblotted using anti- CK17 antibody. High exposure of the immunoblot revealed the presence of cytokeratin 17 in most samples including the samples harvested from naïve mice but with less intensity. The data show 3 separate experiments using with 3 mice per time point for each experiment.