Induction of lung fibrosis in the mouse by intratracheal instillation of fluorescein isothiocyanate is not T-cell-dependent

Abstract

Pulmonary fibrosis is the pathological result of a diverse group of insults. Common features of this group of diseases include chronic inflammation and immune cell activation. The pathogenesis of pulmonary fibrosis is not well defined and the prognosis is poor, highlighting the need for good animal models to elucidate the cellular and molecular events that lead to pulmonary fibrosis. This paper provides insight on a newly described model of pulmonary fibrosis using a single intratracheal challenge with fluorescein isothiocyanate (FITC). Balb-c and C57BL6 mice given intratracheal FITC develop acute lung injury followed by chronic inflammation. Significant increases in lung collagen content compared to saline-treated mice are noted at day 21 after inoculation. T-cell-deficient animals develop similar increases in lung collagen content compared to immunocompetent controls despite the abrogation of specific anti-FITC serum antibodies. Thus, the induction of fibrosis in FITC-challenged mice is not dependent on T cell immunity. Persistent chronic inflammation and acute lung injury may be the inciting events for the development of lung fibrosis in this model.

Figure 1.

Intratracheal FITC causes acute lung injury, chronic persistent inflammation, and pulmonary fibrosis. A single dose of intratracheal FITC was administered to Balb-c mice. Mice were killed at various times and the lungs were examined for histological changes. A: Acute lung injury demonstrated by alveolar wall edema, alveolar exudate, and acute inflammation 1 day after inoculation. B: Dense consolidation with a mixture of both acute and chronic inflammatory cells on day 7. Dense collections of mononuclear cells are present (arrow). C: Residual patchy bronchocentric inflammation at day 21 and increased extracellular matrix. Collections of mononuclear cells persist (arrow). D: Deparaffinized section of lung examined directly with the fluorescent microscope demonstrates persistent FITC staining in the peribronchial areas.

Figure 2.

Increased lung hydroxyproline content after intratracheal FITC. A single dose of intratracheal FITC or PBS was administered to Balb-c or C57BL-6 mice. Mice were killed at day 21 and lungs were assayed for hydroxyproline content. Significant increases in lung hydroxyproline content were demonstrated in Balb-c and C57BL-6 mice. *P < 0.05 compared to same strain PBS control, unpaired t-test; n = 5–8 animals; representative data from seven experiments.

Figure 3.

Increased serum anti-fluorescein IgG after intratracheal FITC. A single dose of intratracheal FITC or PBS was administered to Balb-c or C57BL-6 mice. Mice were killed at day 21 and serum was assayed for fluorescein specific antibodies. Increased production of serum anti-fluorescein IgG was noted in both Balb-c and C57BL-6 mice. *P < 0.05 compared to same strain PBS control, unpaired t-test; n = 5–8 animals; representative data from seven experiments.

Figure 4.

Depletion of CD4 but not CD8 T cell subsets ablate the production of serum anti-fluorescein IgG after intratracheal FITC. Mice were treated with GK1.5 (anti CD4) or 2.43 (anti-CD8) monoclonal antibodies 2 days before and at intervals after a single dose of intratracheal FITC or PBS in Balb-c mice. Mice were killed at day 21 and serum was assayed for fluorescein-specific antibodies. Increased production of serum anti-fluorescein IgG was noted in FITC-treated mice. This production was ablated in CD4-depleted but not CD8-depleted mice. *P < 0.05 compared to PBS treated mice, ANOVA; ^#P < 0.05 compared to FITC-treated mice with normal T cell subsets, ANOVA; n = 5–8 animals; representative data from three experiments.

Figure 5.

Depletion of T cell subsets does not alter lung hydroxyproline content after intratracheal FITC. Mice were treated with GK1.5 (anti CD4) or 2.43 (anti-CD8) monoclonal antibodies 2 days before and at intervals after a single dose of intratracheal FITC in Balb-c mice. Mice were killed at day 21 and lungs were assayed for hydroxyproline content. Depletion of either CD4 or CD8 T cell subsets did not alter the increase in lung hydroxyproline content after intratracheal FITC. *P < 0.05 compared to PBS-treated mice, ANOVA; ^#P not significant compared to FITC-treated mice with normal T cell subsets, ANOVA; n = 18 to 30 animals, pooled data from three identical experiments.

Figure 6.

SCID mice do not develop anti-fluorescein IgG after intratracheal FITC. Balb-c SCID or normal Balb-c mice were treated with a single dose of intratracheal FITC or PBS. Mice were killed at day 21 and serum was assayed for fluorescein-specific antibodies. Increased production of serum anti-fluorescein IgG was noted in normal Balb-c mice but not SCID mice. *P < 0.05 compared to PBS-treated Balb-c mice, ANOVA; ^#P < 0.05; compared to FITC-treated Balb-c mice, ANOVA; n = 5–8 animals.

Figure 7.

SCID mice manifest increased lung hydroxyproline content after intratracheal FITC. Balb-c SCID or normal Balb-c mice were treated with a single dose of intratracheal FITC or PBS. Mice were killed at day 21 and lungs were assayed for hydroxyproline content. Increased hydroxyproline content was noted in both Balb-c and SCID mice treated with FITC compared to PBS-treated controls. There was no difference in lung hydroxyproline content between normal Balb-c and SCID mice treated with FITC. *P < 0.05 compared to PBS-treated same strain control, ANOVA; ^#P not significant compared to FITC-treated Balb-c mice, ANOVA; n = 5–8 animals.

Figure 8.

RAG-KO mice do not develop anti-fluorescein IgG after intratracheal FITC. RAG-KO or normal C57BL-6 mice were treated with a single dose of intratracheal FITC or PBS. Mice were killed at day 21 and serum was assayed for fluorescein-specific antibodies. Increased production of serum anti-fluorescein IgG was noted in normal C57BL-6 mice but not in RAG-KO mice. *P < 0.05 compared to PBS-treated C57BL-6 mice, ANOVA; ^#P < 0.05 compared to FITC-treated C57BL-6 mice, ANOVA; n = 5–8 animals.

Figure 9.

RAG-KO mice manifest increased lung hydroxyproline content after intratracheal FITC. RAG-KO or normal C57BL-6 mice were treated with a single dose of intratracheal FITC or PBS. Mice were killed at day 21 and lungs were assayed for hydroxyproline content. Increased hydroxyproline content was noted in both RAG-KO or normal C57BL-6 mice treated with FITC compared to PBS-treated controls. There was no difference in lung hydroxyproline content between normal RAG-KO or normal C57BL-6 mice treated with FITC. *P < 0.05 compared to same strain PBS control, ANOVA; ^#P not significant compared to FITC-treated normal C57BL-6 mice, ANOVA; n = 5–8 animals.