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. 1998 Dec;153(6):1861-72.
doi: 10.1016/S0002-9440(10)65700-8.

Collagen deposition in a non-fibrotic lung granuloma model after nitric oxide inhibition

C M Hogaboam  1 , C S Gallinat, C Bone-Larson, S W Chensue, N W Lukacs, R M Strieter, S L Kunkel
Affiliations

Collagen deposition in a non-fibrotic lung granuloma model after nitric oxide inhibition

C M Hogaboam et al. Am J Pathol. 1998 Dec.

Abstract

Recent studies support the concept that pulmonary granulomatous inflammation directed by interferon (IFN)-gamma, interleukin (IL)-12, and nitric oxide usually resolves in the absence of fibrosis. To determine whether nitric oxide participates in modulating the fibrotic response during the development of pulmonary granulomas in response to purified protein derivative (PPD), mice presensitized to PPD received daily intraperitoneal injections of N(G)-nitro-D-arginine-methyl ester (D-NAME), N(G)-nitro-L-arginine-methyl ester (L-NAME), or aminoguanidine after delivery of PPD-coated beads to the lungs. Eight days later, morphometric analysis of lung granulomas revealed that L-NAME-treated mice when challenged with PPD in vitro for 36 hours had the largest pulmonary granulomas and the greatest collagen deposition among the treated groups. In addition, equivalent numbers of dispersed lung cells from L-NAME- and aminoguanidine-treated mice produced significantly higher levels of IL-4, monocyte chemoattractant protein (MCP)-1, and macrophage inflammatory protein (MIP)-1alpha and significantly lower levels of eotaxin compared with D-NAME-treated mice. Cultures of dispersed lung cells from L-NAME-treated mice also produced significantly more IL-10 and less IL-12 compared with similar numbers of dispersed lung cells from D-NAME-treated mice. Cultures of isolated lung fibroblasts from L-NAME-treated mice expressed higher levels of C-C chemokine receptor 2 (CCR2) and CCR3 mRNA and contained less MCP-1 and eotaxin protein than a similar number of fibroblasts from D-NAME-treated mice. Thus, nitric oxide appears to regulate the deposition of extracellular matrix in lung granulomas through the modulation of the cytokine and chemokine profile of these lesions. Alterations in the cytokine, chemokine, and procollagen profile of this lesion may be a direct effect of nitric oxide on the pulmonary fibroblast and provide an important signal for regulating fibroblast activity during the evolution of chronic lung disease.

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Figures

Figure 1.
Figure 1.
Composite of PPD-bead granulomas from d-NAME-treated (A), l-NAME-treated (B), and aminoguanidine-treated (C) mice. All mice were maintained on an l-arginine-deficient diet for the duration of the study, and mice received d-NAME, l-NAME, or aminoguanidine for the 8-day PPD-bead challenge period. Histological sections of whole lungs from all three treatment groups on day 8 of the PPD-bead challenge period were subjected to Masson trichrome staining, and collagen content in these pulmonary lesions is highlighted by the light blue coloration. Approximately 95% of the cross-sectional area of the granulomas in the d-NAME-treated mice was occupied by the Sepharose bead alone. Furthermore, PPD-bead granulomas in l-NAME- and aminoguanidine-treated mice contained significantly more collagen than similar granulomas in d-NAME-treated mice (see Table 1 ▶ ).
Figure 2.
Figure 2.
Pulmonary granuloma cross-sectional area (μm2) in mice presensitized to PPD from Mycobacteria, maintained on an l-arginine-deficient diet, and intravenously injected with PPD-coated beads. For 8 days after PPD-bead administration, mice received d-NAME, l-NAME, or aminoguanidine. The dotted line shows the mean size of the Sepharose beads in the pulmonary granulomas. Data shown are means ± SEM from at least three mice. *P ≤ 0.05 compared with d-NAME-treated group.
Figure 3.
Figure 3.
IL-4 (A), IL-10 (B), and IL-12 (C) levels in cell-free supernatants from cultures containing dispersed lung cells from d-NAME-, l-NAME-, and aminoguanidine-treated mice with PPD-bead pulmonary granulomas. All mice were maintained on an l-arginine-deficient diet for the duration of the study, and mice received d-NAME, l-NAME, or aminoguanidine for 8 days after PPD-bead administration. Whole lungs were removed from each group and mechanically dissociated, and 4.0 × 10 dissociated lung cells/well of a six-well tissue culture plate were cultured in the presence of PPD for 36 hours. Data shown are means ± SEM from triplicate wells from each treatment group. *P ≤ 0.05 compared with d-NAME-treated group.
Figure 4.
Figure 4.
Changes in MCP-1 (A), MIP-1α (B), and eotaxin (C) levels in cell-free supernatants from cultures containing dispersed lung cells from d-NAME-, l-NAME-, and aminoguanidine-treated mice with Th1-type pulmonary granulomas. All mice were maintained on an l-arginine-deficient diet for the duration of the study, and mice received d-NAME, l-NAME or aminoguanidine for 8 days after PPD-bead administration. Whole lungs were removed from each group and mechanically dissociated, and 4.0 × 10 dissociated lung cells/well of a six-well tissue culture plate were cultured in the presence of PPD for 36 hours. Data shown are means ± SEM from triplicate wells from each treatment group. *P ≤ 0.05 compared with d-NAME-treated group. τP ≤ 0.05 compared with d-NAME-treated group.
Figure 5.
Figure 5.
RT-PCR analysis of CCR2, CCR3, and procollagen type I and type III mRNA expression in purified lung fibroblasts from d-NAME- and l-NAME-treated mice (A). All mice were maintained on an l-arginine-deficient diet for the duration of the study, and mice received d-NAME, l-NAME, or aminoguanidine for 8 days after PPD-bead administration. Lung fibroblasts were grown out from cultures of dispersed lung cells from day 8 PPD-bead granulomas. Purified fibroblast cultures were left untreated or treated with IL-4 or IFN-γ for 24 hours before mRNA isolation. Fibroblasts from l-NAME-treated exhibited strong CCR2 and CCR3 mRNA expression (see B for densitometry analysis of RT-PCR) but less procollagen I and III expression than fibroblasts from d-NAME-treated mice. However, procollagen gene expression was noted in cultures of fibroblasts from l-NAME-treated mice after exposure of these cells to IFN-γ for 24 hours.
Figure 6.
Figure 6.
MCP-1 (A) and eotaxin (B) levels in cell-free supernatants in cultures of purified lung fibroblasts from d-NAME- or l-NAME-treated mice. All mice were maintained on an l-arginine-deficient diet for the duration of the study, and mice received d-NAME, l-NAME, or aminoguanidine for 8 days after PPD-bead administration. Lung fibroblasts were grown out from cultures of dispersed lung cells from day 8 PPD-bead granulomas. Purified fibroblasts cultures were left untreated or treated with IL-4 or IFN-γ for 24 hours before removal of cell-free supernatants for ELISA. Cultures of lung fibroblasts from l-NAME-treated mice with PPD-bead granulomas generated significantly less MCP-1 after cytokine activation than similar numbers of fibroblasts derived from d-NAME-treated mice (A). Similarly, less eotaxin was present in cultures of fibroblasts from l-NAME-treated mice than cultures of fibroblasts from d-NAME-treated mice (B). Data shown are means ± SEM of three separate experiments.
Figure 7.
Figure 7.
TGF-β levels in cell-free supernatants from cultures of fibroblasts isolated from d-NAME- or l-NAME-treated mice. All mice were maintained on an l-arginine-deficient diet for the duration of the study, and mice received d-NAME, l-NAME, or aminoguanidine for 8 days after PPD-bead administration. Lung fibroblasts were grown out from cultures of dispersed lung cells from day 8 PPD-bead granulomas. Purified fibroblasts cultures were left untreated or treated with IL-4 or IFN-γ for 24 hours before removal of cell-free supernatants for ELISA. Cultures of lung fibroblasts from l-NAME-treated mice with PPD-bead granulomas generated significantly more TGF-β after IL-4 activation than similar numbers of fibroblasts derived from d-NAME-treated mice. Data shown are means ± SEM of three separate experiments.
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