Induction of prolonged infiltration of T lymphocytes and transient T lymphocyte–dependent collagen deposition in mouse lungs following adenoviral gene transfer of CCL18

Abstract

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 enzymelinked 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 ␤1 (TGF␤1) or interleukin-13, although active TGF␤1 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 TGF␤1 and collagen production in T lymphocyte/fibroblast cocultures.Conclusion. CCL18 promotes selective, long-term pulmonary infiltration of T lymphocytes and infiltration-dependent accumulation of collagen through a TGF␤1-dependent mechanism.

Splice isoforms of human interleukin-4 are functionally active in mice in vivo

“…23 A relative increase in IL-4 and a decrease in IL-4d2 have been associated with better survival in patients with severe sepsis. 24 In this study, we used a replication-deficient adenovirus system [25][26][27] for gene delivery to mouse lung to overexpress human IL-4 or IL-4d2 in vivo. We show that hIL-4 and hIL-4d2 partially share functional activities in mice in vivo with their species-specific counterparts, but they differ from each other in that over-expression of hIL-4d2 appears to have a more pronounced pro-inflammatory effect.…”

“…[25][26][27] Briefly, GenBank consensus sequences for these cytokines were used to artificially synthesize (GenScript, Piscataway, NJ) the DNA fragments corresponding to the respective cDNAs. The fragments were subcloned into a shuttle vector, and transferred into a recombinant replication-deficient adenovirus vector (AdV) using RAPAD Ò technology (ViraQuest, North Liberty, IA).…”

“…The adenoviruses were instilled intra-tracheally as previously described in detail. [25][26][27] Fluorescence microscopy of unstained lung sections for GFP was used to confirm infectivity in vivo. Quantitative PCR of lung homogenates was used to confirm over-expression of mRNAs for the delivered cytokines.…”

“…Bronchoalveolar lavage (BAL), pulmonary homogenates, histological and immunohistological analyses, and flow cytometry were all performed as previously described. [25][26][27] Multiplex analyses (Luminex, Austin, TX) of cytokine levels in BAL and lung homogenates were performed in triplicate in each sample from each animal, and the mean values and standard deviations were calculated for the triplicates.…”

“…Infectivity of the viruses, including AdV-NULL, was evident from GFP fluorescence of the infected cells at 24-72 hr after the virus had been added to the cultures, similar to a previous report (see figure 1a in ref. 25). Delivery of the encoded genes was confirmed by RT-PCR with specific primers.…”