THE STIMULATING EFFECT OF INTERLEUKIN-33 ON PROLIFERATION OF THE PRIMARY HUMAN LUNG FIBROBLASTS

Interleukin (IL)-33 is a multifunctional cytokine that belongs to the IL-1 cytokine family and expressed by multiple organs and cell types. Recent studies have showed that IL-33 plays an etiological role in several fibrotic disorders and may be involved in the pathogenesis of chronic respiratory diseases. It has been reported that IL-33–induced cutaneous fibrosis is associated with the increased fibroblast proliferation and altered expression of extracellular matrix-modifying genes. However, the role of IL-33 in regulating of functions of lung fibroblasts remains unclear. In the present study we examined the effect of IL-33 on proliferation of human lung fibroblasts. Five primary lines of normal adult human lung fibroblasts were cultured for 3-7 days in the presence of increasing concentrations of IL-33. We have observed that normal human lung fibroblasts responded in a dose-dependent manner to treatment with recombinant human IL-33 by increasing proliferation rates 1.5to 2.3 fold compared to the non-stimulated control. The maximum effect of IL-33 on fibroblast proliferation was observed in the cytokine concentrations range from 2 ng/ml to 100 ng/ml. These results suggest that IL-33 may play an important role in the regulation of the human lung fibroblast proliferation. Human lung fibroblasts activated by IL-33 may act as effector cells not only in the pathogenesis of lung diseases, but also in lung remodeling processes.

I nterleukin (IL)-33 is a multifunctional cytokine that belongs to the IL-1 cytokine family and mainly expressed by fibroblasts, epithelial cells, endothelial cells, and smooth muscle cells in the lung, kidney, skin, stomach and central nervous system [2,4,10]. In the absence of pro-inflammatory stimuli, IL-33 localizes to the nucleus where in its uncleaved form it interacts with histones H2A and H2B [9]. IL-33 is released from cells undergoing necrotic cell death and thus functions as a damage-associated molecular pattern (DAMP) [11]. The biological effects of IL-33 are mediated through interaction with the receptors ST2 and IL-1 Receptor Accessory Protein, both of them are widely expressed by fibroblasts, mast cells, macrophages, innate immune cells and T-helper 2 (Th2) cells [3]. Circulating IL-33 is elevated in patients with pulmonary diseases, anaphylaxis, rheumatoid arthritis, atherosclerosis, Alzheimer's disease, inflammatory bowel disease and sepsis [1]. Recent studies have revealed a connection between IL-33/ST2 and development of fibrotic dis-orders, such as pulmonary fibrosis, scleroderma and progressive systemic sclerosis [6,7,8,11]. It has been reported that IL-33-induced cutaneous fibrosis is associated with the increased fibroblast proliferation and altered expression of extracellular matrixmodifying genes [5]. However, the role of IL-33 in regulation of functions of lung fibroblasts remains unclear.
The aim of the present study was to investigate the effect of rhIL-33 on proliferation of human lung fibroblasts in vitro.

Materials and Methods
Five primary normal adult human lung fibroblasts lines (NHLF1-NHLF5) were purchased from NIH (Bethesda, MD) and Lonza Walkersville (Walkersville, MD). Fibroblast lines were grown in T75 culture flasks in a humidified atmosphere of 5% CO 2 at 37°C in the high serum tissue culture medium DMEM with glutamine, sodium pyruvate, antibiotic/antimycotic, and 10% bovine calf serum.
NHLF were tested in passage three-seven. Cells were grown to confluency, treated by trypsinization, washed, and replaced in the high serum tissue culture medium at 2x10 3 cells per well in 96-well flat-bottom tissue culture plates. After overnight incubation in the high serum tissue culture medium the medium in each well was replaced with RPMI 1640 containing all supplements, except the serum concentration was decreased to 0.5% (the low serum tissue culture medium). The fibroblasts were incubated for another 24 h before adding the test substances. Recombinant human IL-33 (R&D Systems) was used in the concentrations of 1, 2, 10, 50, 100 and 300 ng/ml. The low serum tissue culture medium alone was the negative control. Proliferation of fibroblasts was analyzed using the cell proliferation assay (CellTiter Aqueous; Promega) in accordance with the manufacturer's recommendations, after the fibroblasts were incubated with the test substances for 3-7 days. Changes in the cell proliferation rates were assessed in quintuplicate.

Results and Discussion
Normal human primary pulmonary fibroblasts proliferate in response to stimulation with IL-33. One-way ANOVA revealed a significant effect of IL-33 (in the concentrations of 2 to 300 ng/ml) on the fibroblast proliferation. Increase in the proliferation rate was observed in four lines ( Fig.).
One cell line did not respond by a change in proliferation to treatment with IL-33 (data are not shown). The NHLF responded in a dose-dependent manner to treatment with recombinant human IL-33, by increasing proliferation rates 1.5 to 2.3 fold compared to the non-stimulated control fibroblasts. The maximum effect of IL-33 on the fibroblast proliferation was observed in the cytokine concentration of 2 ng/ml with a gradual decline in the concentrations of 100 ng/ml and 300 ng/ml. The average increase in proliferation rates in this concentration was 1.95 ± 0.25-fold compared with the non-stimulated control cultures.
The results obtained suggest that IL-33 may play an important role in the regulation of the human lung fibroblast proliferation.
CONCLUSIONS Recombinant human interleukin-33 stimulates the primary human lung fibroblasts proliferation in a dose-dependent manner.
Human lung fibroblasts activated by IL-33 may act as effector cells not only in the pathogenesis of lung diseases, but also in lung remodeling processes.