Effect of IL-2-Bax, a novel interleukin-2-receptor-targeted chimeric protein, on bleomycin lung injury

Abstract

The role of lymphocytes in the pathogenesis of lung fibrosis is not clear, but the weight of the evidence supports a pro-fibrotic effect for lymphocytes. The high-affinity interleukin-2 receptor (haIL-2R) is expressed on activated, but not quiescent, T lymphocytes. This selective expression of haIL-2R provides the basis for therapeutic strategies that target IL-2R-expressing cells. We hypothesized that elimination of activated lymphocytes by IL-2R-targeted chimeric proteins might ameliorate lung fibrosis. We investigated the effects of IL-2-Bax, a novel apoptosis-inducing IL-2R-targeted chimeric protein, on bleomycin-induced lung injury in mice. Treatment groups included (i) a single intratracheal instillation of bleomycin and twice-daily intraperitoneal injections of IL-2-Bax; (ii) intratracheal bleomycin and intraperitoneal IL-2-PE66(4Glu), an older-generation chimeric protein; (iii) intratracheal bleomycin/intraperitoneal PBS; (iv) intratracheal saline/intraperitoneal PBS. Lung injury was evaluated 14 days after intratracheal instillation by cell count in bronchoalveolar lavage (BAL) fluid, semi-quantitative and quantitative histomorphological measurements and by biochemical analysis of lung hydroxyproline. Bleomycin induced a BAL lymphocytosis that was significantly attenuated by IL-2-Bax and IL-2-PE66(4Glu). However, morphometric parameters and lung hydroxyproline were unaffected by the chimeric proteins. These results show that IL-2-Bax reduces the lymphocytic infiltration of the lungs in response to bleomycin, but this effect is not accompanied by a decrease in lung fibrosis.

Figure 1

IL-2-PE66^4Glu dose-dependently inhibits protein synthesis in lung cells from bleomycin-treated mice. Cells were extracted from the lungs of mice 6 days after intratracheal instillation of bleomycin, and cultured ex vivo. The effect of different concentrations of IL-2-PE66^4Glu on protein synthesis (³H-leucine uptake) was measured. Median values of measurements done in triplicate are showed. The experiment was performed twice.

Figure 2

IL-2-Bax and IL-2-PE66^4Glu attenuated the BAL lymphocytosis induced by bleomycin. Experimental groups and treatment protocols are detailed in the Materials and Methods section. Mice were studied 14 days after intratracheal instillation. Mean + SEM values (10⁶ cells/ml) are showed for BAL lymphocyte counts, and were compared by means of anova/Bonferroni test.

Figure 3

Changes in lung cell (LC) subpopulations induced by IL-2-Bax. All animals received intratracheal bleomycin on day 0. Twice-daily injections of IL-2-Bax or PBS (three mice per group) were commenced 12–16 h later, and continued until the evening before killig, for a total of 6 days. LC were extracted on day 7. Percentages of (a) CD3+ and (b) CD8+ cells are showed, as detected by means of flow cytometry. Each point represents cells from a single animal. Comparison between the two groups was by the Mann–Whitney test.

Figure 4

Morphological and biochemical measurements of lung injury. Experimental groups and treatment protocols are detailed in the Materials and methods section. Mice were studied 14 days after intratracheal instillation. (a) Semi-quantitative morphological index (SMI) – each point represents the score for a single animal; horizontal lines represent median scores; comparison between groups was by the Kruskal–Wallis test; (b) alveolar wall area (mean + SEM, comparison between groups by anova/Bonferroni test); (c) fibrosis fraction (mean + SEM, anova/Bonferroni test); (d) lung hydroxyproline (mean + SEM, anova/Bonferroni test). IL-2-Bax and IL-2-PE66^4Glu did not affect bleomycin -induced fibrosis, as assessed by these parameters.