Metalworking fluid with mycobacteria and endotoxin induces hypersensitivity pneumonitis in mice

Abstract

Background: Human cases of hypersensitivity pneumonitis (HP) have been reported among machinists for over 10 yr. Although mycobacteria have been implicated as causal agents, this has not been established in experimental studies and the mechanisms remain unclear. Other constituents of in-use metalworking fluids (MWFs) may also contribute to the development of lung disease. We investigated the potential for Mycobacterium immunogenum (MI) in MWFs to induce HP.

Methods: Mice were exposed intranasally for 3 wk to MI (isolated from MWFs), Saccharopolyspora rectivirgula (positive control), saline, endotoxin, MWFs spiked with endotoxin and/or MI, used MWFs, and particulate-fortified used MWFs. Responses were assessed 96 h after the last exposure.

Results: Mice exposed to MI in MWFs developed lung pathology consistent with HP along with significantly more monocytes and neutrophils in lung lavage, increased CD4+/CD8+ T-lymphocyte ratio, and marked pulmonary lymphocytosis on histologic examination when compared with saline-treated control mice. Mice with Grade 2 or higher pathology (0-4 point scale) exhibited significantly elevated macrophage inflammatory protein-1alpha and IL-10 and a trend toward higher RANTES 96 h after the final dose. Endotoxin coexposure augmented lung pathology.

Conclusion: MWFs containing mycobacteria induced granulomatous lung lesions, peribronchiolar lymphocytosis, increased cell concentrations in lavage, and up-regulation of several cytokines. These findings are consistent with HP.

Figure 1.

Micrographs of lung sections stained with Masson's trichrome. Control group: mice exposed to saline alone with no sign of hypersensitivity pneumonitis (HP) (A, B); expression of HP with fibrosis in mice exposed to Saccharopolyspora rectivirgula (positive control for HP) (C, D). The areas indicated by the box in the low-magnification figures are shown at higher magnification in the micrographs on the right.

Figure 2.

Results of bronchoalveolar lavage fluid analyses. Values are expressed as mean and SE. *p < 0.05, **p < 0.01, and ***p < 0.001 as compared with mice exposed to saline (t test for equal and unequal variances). (A) Differential cells counts. (B) Cytokines. (C) Cytokines and chemokines.

Figure 2.

Results of bronchoalveolar lavage fluid analyses. Values are expressed as mean and SE. *p < 0.05, **p < 0.01, and ***p < 0.001 as compared with mice exposed to saline (t test for equal and unequal variances). (A) Differential cells counts. (B) Cytokines. (C) Cytokines and chemokines.

Figure 3.

Percentage of experimental groups yielding lung histopathology grade 2 or higher (gray bars) or Grade 3 or higher (black bars). Mean histology grade of negative control mice (saline) was significantly different from the groups MWF0+MI (p < 0.05), MI and MWF-UF (p < 0.01), and MWF0+MI+ENDO (p < 0.001). A t test was used for comparison.

Figure 4.

CD4⁺/CD8⁺ T-lymphocyte ratio (mean and SE) in the bronchoalveolar lavage fluid of exposed and control mice. Mean of negative control mice was significantly different from the groups MWF 0+MI (*p < 0.05), SR, MWF-U and MWF-UF (**p < 0.01), and MI (***p < 0.001). A t test was used for comparison.

Figure 5.

Micrographs of lung sections (stained with Masson's trichrome) from mice exposed to used MWFs (A, B, C) and lung sections (hematoxylin and eosin staining) from mice exposed to used fortified MWFs (D, E, F). The areas indicated by the box in the low-magnification figures are shown at higher magnification in the micrographs. Granulomas and fibrosis (see blue staining) representing chronic forms of HP and giant cells (arrow) developed in the lungs exposed to used MWF samples (MWF-U and MWF-UF).