Faculty Bibliography

Phagocytic clearance of apoptotic cells by macrophages is an essential part in the resolution of inflammation. It coincides with activation of repair mechanisms, including accumulation of extracellular matrix. A possible link between clearance of apoptotic debris and accumulation of extracellular matrix has not been investigated. Production of collagen was measured in primary fibroblasts cocultured with macrophages. Ingestion of apoptotic cells by monocyte-derived macrophages led to up-regulation of collagen. Direct contact between macrophages and fibroblasts was not required for collagen up-regulation. Macrophages produced TGF-beta following ingestion of apoptotic cells, but the levels of this cytokine were lower than those required for a significant up-regulation of collagen. Simultaneously, the levels of TGF-beta-induced (TGFBI), or keratoepithelin/BIGH3, mRNA and protein were increased. In contrast, primary alveolar macrophages stimulated collagen production without exposure to apoptotic cells; there was no further increase in the levels of TGFBI, mRNA or protein, or collagen after ingestion of apoptotic cells. Stimulation of fibroblasts with TGFBI down-regulated MMP14 levels, decreased DNA binding by p53, increased DNA binding by PU.1, and up-regulated collagen protein but not mRNA levels. Overexpression of MMP14 or p53, or small interfering RNA-mediated inhibition of PU.1 led to an increase in MMP14 and a decline in collagen levels, whereas small interfering RNA-mediated inhibition of MMP14 led to elevation of collagen levels. In conclusion, monocyte-derived but not alveolar macrophages produce TGFBI following ingestion of apoptotic cells, leading to the down-regulation of MMP14 levels in fibroblasts through a mechanism involving p53 and PU.1, and to subsequent accumulation of collagen.

Elevated pulmonary levels of CCL18 have been associated with influx of T lymphocytes, collagen accumulation, and a decline in lung function in pulmonary fibrosis patients. We previously reported that overexpression of CCL18 in mouse lungs triggers selective infiltration of T lymphocytes and moderate lymphocyte-dependent collagen accumulation. We hypothesized that in combination with bleomycin injury, overexpression of CCL18 will worsen the severity of lung inflammation and fibrosis. Mice were infected with a replication-deficient adenovirus encoding CCL18 and then instilled with bleomycin; control mice were challenged with either CCL18 overexpression or bleomycin. Additive effects of CCL18 overexpression and bleomycin injury were observed on pulmonary inflammation, particularly on T-cell infiltration, and increased levels of tumor necrosis factor-alpha, interferon-gamma, matrix metalloproteinase (MMP)-2, and MMP-9. Despite the additive effect on inflammation, CCL18 overexpression unexpectedly attenuated the bleomycin-induced collagen accumulation. Pulmonary levels of active transforming growth factor-beta1 mirrored the changes in collagen levels. Depletion of T cells with antilymphocyte serum or pharmacological inhibition of MMPs with GM6001 abrogated accumulation of collagen and increases in the levels of tumor necrosis factor-alpha, interferon-gamma, and active transforming growth factor-beta1. Thus, CCL18-stimulated T-lymphocytic infiltration is by itself mildly profibrotic to a healthy lung, whereas it partially protects against lung fibrosis in an inflammatory profibrotic pulmonary milieu.

A CC chemokine, CCL18, has been previously reported to stimulate collagen production in pulmonary fibroblasts. This study focused on the role of protein kinase C (PKC) in the profibrotic signaling activated by CCL18 in pulmonary fibroblasts. Of the three PKC isoforms that are predominantly expressed in fibroblasts (PKCalpha, PKCdelta, and PKCepsilon), two isoforms (PKCdelta and PKCepsilon) have been implicated in profibrotic intracellular signaling. The role of PKCalpha-mediated signaling in the regulation of collagen production remains unclear. In this study, PKCalpha was found mostly in the cytoplasm, whereas PKCdelta and PKCepsilon were found mostly in the nucleus of cultured primary pulmonary fibroblasts. In response to stimulation with CCL18, PKCalpha but not PKCdelta or PKCepsilon underwent rapid (within 5-10 min) transient phosphorylation and nuclear translocation. Inhibition with dominant-negative mutants of PKCalpha and ERK2, but not PKCdelta or PKCepsilon, abrogated CCL18-stimulated ERK2 phosphorylation and collagen production. The effect of CCL18 on collagen production and the activity of collagen promoter reporter constructs were also abrogated by a selective pharmacologic inhibitor of PKCalpha Gö6976. Stimulation of fibroblasts with CCL18 caused an increase in intracellular calcium concentration. Consistent with the known calcium dependence of PKCalpha signaling, blocking of the calcium signaling with the intracellular calcium-chelating agent BAPTA led to abrogation of PKCalpha nuclear translocation, ERK2 phosphorylation, and collagen production. These observations suggest that in primary pulmonary fibroblasts, PKCalpha but not PKCdelta or PKCepsilon mediate the profibrotic effect of CCL18. PKCalpha may therefore become a viable target for future antifibrotic therapies.

OBJECTIVE:Levels of CCL18 are elevated in patients with scleroderma lung disease and other fibrotic pulmonary diseases associated with T lymphocyte involvement. We sought to determine whether CCL18 alone can induce pulmonary T lymphocytic infiltration and fibrosis in mouse lungs. METHODS:An adenovirus vector was constructed and used for CCL18 delivery to mouse lungs in vivo. Immunohistochemical, flow cytometric, and enzyme-linked immunosorbent assay analyses were used to assess the resulting changes. RESULTS:Overexpression of CCL18 led to massive perivascular and peribronchial infiltration of T lymphocytes. Although the expression of CCL18 peaked on day 7, the infiltration persisted up to day 64 after infection. The infiltrates were negative for proliferating cell nuclear antigen and TUNEL, suggesting the role of cell trafficking, rather than proliferation and apoptosis, in the infiltration dynamics. Patchy destruction of the alveolar architecture and collagen accumulation in association with the infiltrates were also noticed. These changes were infiltration-dependent, rather than CCL18-dependent, since treatment with antilymphocyte serum completely abrogated the CCL18-induced changes. The infiltrates consisted almost exclusively of T lymphocytes that were minimally activated, with a minimal increase in the expression of CD69 and no changes in the expression of CD25, Fas, FasL, or CD40L. There was no increase in total pulmonary levels of profibrotic cytokines transforming growth factor beta1 (TGFbeta1) or interleukin-13, although active TGFbeta1 was present locally in association with the infiltrates and areas of distorted alveolar architecture. Prestimulation of primary T lymphocytes with CCL18 in vitro caused an up-regulation of TGFbeta1 and collagen production in T lymphocyte/fibroblast cocultures. CONCLUSION/CONCLUSIONS:CCL18 promotes selective, long-term pulmonary infiltration of T lymphocytes and infiltration-dependent accumulation of collagen through a TGFbeta1-dependent mechanism.