Novel Functions of Intracellular IL-1ra in Human Dermal Fibroblasts: Implications in the Pathogenesis of Fibrosis  Siva Kanangat, Arnold E. Postlethwaite, Gloria C. Higgins, Karen A. Hasty  Journal of Investigative Dermatology  Volume 126, Issue 4, Pages 756-765 (April 2006) DOI: 10.1038/sj.jid.5700097 Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Overexpression of icIL-1ra in HF-icIL-1ra. (a) Equal number of cells (HF-icIL-1ra and HF-Vector) were stimulated with 0 and 1.0ng of hrIL-1β or 10ng/ml of hrTNF-α. After 24hours, total RNA was extracted and mRNA levels of icIL-1ra and GAPDH were estimated by real-time RT-PCR. Ratios of icIL-1ra type 1 to GAPDH messages are plotted in the graph. (b) Equal numbers of HF-icIL-1ra and HF-Vector maintained in complete DMEM for 48hours were harvested and lysed. The clarified cell lysates were tested for icIL-1ra type 1 by ELISA. The error bars indicate mean±SD of three separate experiments on the same batch of stably transfected fibroblasts. Journal of Investigative Dermatology 2006 126, 756-765DOI: (10.1038/sj.jid.5700097) Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Myofibroblast morphology of HF-icIL-1ra. (a) HF-Vector with normal spindle-shaped fibroblast morphology after culturing in complete DMEM for 6 weeks with medium changes every 5 days. (b) HF-icIL-1ra with myofibroblast-like morphology. (c, d) Cells were fixed and permeabilized (Cytofix/Cytoperm) and were subjected to immunoperoxidase staining with mouse anti-human α-SMA (Sigma) clone 1A 4 monoclonal antibody followed by color reaction developed by a streptavidin–horseradish peroxidase system as described. (c) HF-Vector with little α-SMA staining. (d) HF-icIL-1ra with α-SMA staining. (e) HFF-TGF-passed cells with myofibroblast-like morphology. (f) HFF-TGF with intense staining for α-SMA. Journal of Investigative Dermatology 2006 126, 756-765DOI: (10.1038/sj.jid.5700097) Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Enhanced levels of α-SMA and PAI mRNA in HF- icIL-1ra. HF-icIL-1ra and HF-Vector were maintained in complete DMEM for 6 weeks with medium changes every 5 days. Cells were harvested and total cellular RNA was extracted and reverse transcribed. The cDNA thus obtained was amplified by real-time PCR and quantified by SYBR Green using α-SMA primers and PAI primers (Table 4). The values are expressed as ratios of Ct values of α-SMA to those of the housekeeping gene GAPDH. The values indicate mean±SD of three independent experiments performed on the same batch of stably transfected fibroblasts. Journal of Investigative Dermatology 2006 126, 756-765DOI: (10.1038/sj.jid.5700097) Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Reduced mRNA in HF-icIL-1ra. HF-icIL-1ra and HF-Vector were maintained in complete DMEM for 6 weeks with medium changes every 5 days. Cells were harvested in Tri-Reagent and total cellular RNA was extracted. One microgram of total RNA was reverse transcribed in a 20μl reverse transcription reaction mixture and 5.0μl of the cDNA was amplified by 28 cycles of PCR using primers specific for GAPDH and MMP-1. The PCR products were analyzed on a 2% agarose gel, stained with ethidium bromide, and photographed. Journal of Investigative Dermatology 2006 126, 756-765DOI: (10.1038/sj.jid.5700097) Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Reduced expression of IL-1- and TNF-induced MMP- 1mRNA in HF-icIL-1ra. HF-icIL-1ra and HF-Vector were stimulated with 1.0ng/ml IL-1β or10ng/ml TNF-α for 12–16hours. MMP-1 and GAPDH message levels were estimated using real-time RT-PCR. Three separate experiments were performed on the same batch of stably transfected fibroblasts. The results are represented as the reciprocal of the ratios of the Ct values of MMP-1 to those of GAPDH (the housekeeping gene). Journal of Investigative Dermatology 2006 126, 756-765DOI: (10.1038/sj.jid.5700097) Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 Reduced levels of MMP-1 protein in HF-icIL-1ra type 1. HF-icIL-1ra and HF-Vector were cultured for 48hours with hrIL-1β (1.0ng/ml) or hrTNF-α (5ng/ml) and MMP-1 protein secreted into the culture medium was measured by ELISA. Journal of Investigative Dermatology 2006 126, 756-765DOI: (10.1038/sj.jid.5700097) Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 7 Normal expression of TIMP-1 mRNA in fibroblasts overexpressing icIL-1ra. HF-icIL-1ra type 1 HF-icIL-1ra and HF-Vector were stimulated with 0 or 10ng/ml TNF-α for 12–16hours. Total cellular RNA was extracted and TIMP-1 and GAPDH message levels were estimated using SYBR Green real-time RT-PCR. The values are expressed as the reciprocal ratios of Ct values of TIMP-1 mRNA and collagen type I mRNA to those of the housekeeping gene GAPDH. The values indicate mean±SD of three independent experiments performed on the same batch of stably transfected fibroblasts. Journal of Investigative Dermatology 2006 126, 756-765DOI: (10.1038/sj.jid.5700097) Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 8 Transfection of HF-icIL-1ra with antisense oligonucleotide directed toward icIL-1ra restores IL-1-induced MMP-1 mRNA expression. Phosphorothioate-derivatized antisense oligodeoxynucleotide complimentary to −6 to +12 of the natural icIL-1ra was synthesized and oligonucleotide with a scrambled sequence was prepared by a similar method as a control. HF- icIL-1ra stimulated with 1.0ng/ml IL-1β was transfected with 300mM of antisense icIL-1ra type 1 oligonucleotide (24hours prior to stimulation with 1.0ng/ml of IL-1β) using LipofectAMINE. PBS and LipofectAMINE alone served as additional controls. The cells were harvested 12–18hours after stimulation for RNA extraction. Total RNA was reverse transcribed and MMP-1 mRNA was estimated by real-time RT-PCR. The values indicate mean±SD of three independent experiments performed on the same batch of transfected geneticin-selected fibroblasts (PBS: PBS alone; LIPO: LipofectAMINE alone; ANTIS-LIP: antisense icIL-1ra oligonucleotide+LipofectAMINE; SCRAM-LIP: scrambled oligonucleotide+LipofectAMINE). Journal of Investigative Dermatology 2006 126, 756-765DOI: (10.1038/sj.jid.5700097) Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions