Induction of chemokines by low-dose intratracheal silica is reduced in TNFR I (p55) null mice

Abstract

Previous studies suggest that tumor necrosis factor alpha (TNF-alpha) and the TNFRI (p55) and TNFRRII (p75) receptors mediate the pulmonary fibrotic response to silica. In order to further define the role of the TNFRI (p55) receptor in induction of profibrotic chemokines by low-dose silica/crystalline silica (50 micro g/50 micro l/mouse) or control diluent saline was instilled into the trachea of TNFRI gene ablated ((-/-)) and C57BL/6 (WT) control mice. Lung tissue was harvested and bronchoalveolar lavage (BAL) performed 24 h and 28 days following silica administration. Selected profibrotic chemokine mRNAs were quantified by ribonuclease protection assay, normalized to ribosomal protein L32 mRNA content and expressed relative to saline control treated lungs. Induction of MIP-1beta, MIP-1alpha, MIP-2, IP-10, and MCP-1 mRNAs was attenuated in the TNFRI(-/-) mice, in comparison to WT mice, particularly at 28 days after exposure. ELISA assays for MIP-1alpha and MIP-2 in homogenized lung tissue similarly demonstrated marked induction of both chemokines 24 h after silica treatment, which was persistent at 28 days in WT but not in TNFRI(-/-) mice. The percentage of BAL cells that was neutrophils was comparably increased in WT and RI(-/-) lungs at 24 h (49 +/- 12% vs. 46 +/- 10%) and 28 days (6.2 +/- 1.5% vs. 4.5 +/- 1%). The increase in total lavagable cells and BAL protein was also independent of strain. Histology revealed mild alveolitis without granuloma formation in both strains, slightly decreased in TNFRI(-/-). This study demonstrates an increase in pro-fibrotic chemokines in response to a single intratracheal exposure to crystalline silica that was sustained at 28 days after treatment in WT but not in TNFRI(-/-) mice. Silica dependent recruitment of neutrophils to the alveolar space and alveolar protein leak were, however, not altered by the absence of the TNF receptor.

FIG. 1

TNFRI-dependent induction of chemokine mRNAs by intratracheal silica. (A) Representative phosphorimage of chemokine ribonuclease protection assay (RPA) of total left lung RNA (5 µg), harvested from C57BL/6 (WT) and TNFRF^−/− mice 24 h and 28 days after intratracheal silica (50 µg/mouse) or control saline treatment. (P) Undigested, radiolabeled RPA template set, utilized as size markers; (+) positive control RNA; and (−) negative yeast control RNA. The templates are 29 nucleotides larger than the fragments (– –) protected by the mRNAs of interest. (B) Cumulative data: chemokine mRNA levels in WT (black bars) and TNFRF^−/− mice (hatched bars) relative to levels in saline-treated lungs. Mean ± SEM, n = 3–5, *p ≤ 0.05 WT versus TNFRI^−/− at same time point.

FIG. 2

Quantitation of MIP-1α gene expression in lungs of wild-type or TNFRI^−/− mice following, it, silica (50 µg/mouse). (A) MIP-1α mRNA measured by RPA and normalized to rpL32 mRNA content of each sample. (B) MIP-1α protein content of left lung homogenate measured by ELISA and normalized to total protein of each sample of C57BL/6 (WT) and TNFRI^−/− mice, 24 h and 28 days after intratracheal saline (black bars) or silica (hatched bars); n = 4–5 mice for each strain and treatment, mean ± SEM. *p ≤ 0.02, saline versus silica-treated from same strain; #p ≤ 0.01, silica-treated WT versus TNFRI^−/− at same time point.

FIG. 3

Quantitation of MIP-2 gene expression in lungs of wild-type or TNFRI^−/− mice following intratracheal silica (50 µg/mouse). (A) MIP-2 mRNA measured by RPA and normalized to rpL32 mRNA content of each sample. (B) MIP-2 protein content of left lung homogenate measured by ELISA and normalized to total protein of each sample of C57BL/6 (WT) and TNFRF^−/− mice, 24 h and 28 days after intratracheal saline (black bars) or silica (hatched bars); n = 4–5 mice for each strain and treatment; mean ± SEM. *p ≤ 02, saline versus silica-treated of same strain; #p ≤ 01, silica-treated WT versus TNFRI^−/− at same time point.

FIG. 4

Histopathology in C57BL/6 and TNFRI^−/− mouse lung following intratracheal silica. Representative lung sections taken from C57BL/6 and TNFRI^−/− mice at (A) 24 h and (B) 28 days after intratracheal saline or silica (50 µg/mouse) treatment, stained by standard hematoxylin & eosin. (C) Lung injury and fibrosis score assigned by independent observer (R.B.) based on light microscopic assessment of degree of abnormality in alveoli, bronchioles, and bronchi as described in Materials and Methods.

FIG. 5

Cell count and differential of bronchoalveolar lavage of C57BL/6 (WT) and TNFRI^−/− mice following intratracheal silica (50 µg/mouse). The cell pellets from the first two ml of BAL collected from C57BL/6 (WT) and TNFRI^−/− mice 24 h and 28 days after intratracheal saline (black bars) or silica (hatched bars) was analyzed for (A) cell count by hemocytometer and (B–C) percent macrophages, neutrophils, and lymphocytes by Diff-Quick stain and light microscopic analysis of nuclear morphology of cytospin preparations; n = 4 mice/strain, time, and treatment.

FIG. 6

Total protein and LDH content of bronchoalveolar lavage of C57BL/6 (WT) and TNFRI^−/− mice following intratracheal silica. Mice were lavaged (1 ml × 10) 24 h and 28 days after intratracheal saline (black bars) or silica (hatched bars). Recovered lavage from the first two aliquots from each animal was pooled, centrifuged to remove cells and debris, and analyzed for total protein concentration and for lactate dehydrogenase content; *p ≤ 0.04 saline versus silica treated from same strain; no significant difference was detected at either time point between silica-treated WT versus TNFRI^−/− mice.