Counterregulation of Interleukin-18 mRNA and Protein Expression During Cutaneous Wound Repair in Mice

Abstract

Recent work has suggested interleukin-18 to represent a proinflammatory cytokine that contributes to systemic and local inflammation. As the process of cutaneous wound healing crucially involves an inflammatory phase of repair, we investigated the regulation of interleukin-18 during the repair process. In non-wounded skin we observed high levels of interleukin-18 mRNA, whereas corresponding interleukin-18 protein was expressed only at low basal levels. Upon injury, we found a rapid and large induction of interleukin-18 protein expression, which is directly correlated with decreasing mRNA levels within the wound. Immunohistochemical analysis revealed different sites of expression in the wounded area, with keratinocytes as one major source of interleukin-18 production. The counterregulation of interleukin-18 mRNA and protein expression during wound repair in vivo might represent a general mechanism for interleukin-18 expressional regulation, as cytokine-stimulated keratinocytes exhibit a similar downregulation of interleukin-18 mRNA that is directly associated with increasing interleukin-18 protein levels in vitro. The rapid induction of interleukin-18 during wound healing suggests a role for interleukin-18 within the early phase of repair rather than a role in costimulation of interferon-gamma release from T cells, which are present in high numbers within the wounded area only during the late inflammatory phase of repair.

Leptin and Wound Inflammation in Diabeticob/obMice

“…RNA isolation and RNase protection assays were carried out as described previously (18,19 (17,20). Total protein (10 -50 g diluted in lysis buffer [20] to a final volume of 50 l) from skin lysates was subsequently analyzed for the presence of immunoreactive MIP-2, MCP-1, IL-1␤, and TNF-␣ by enzymelinked immunosorbent assay (ELISA) using the Quantikine murine ELISA kits (R&D systems, Wiesbaden, Germany). Fifty microliters of supernatants from control and cytokine (2 nmol/l IL-1␤, 2 nmol/l TNF-␣)-stimulated murine PAM 212 keratinocytes were analyzed for MIP-2 by ELISA (R&D systems, Wiesbaden, Germany).…”

“…Cytokines were from Roche (Mannheim, Germany). Scab lysates were prepared in lysis buffer (20) and given to quiescent keratinocytes in a final concentration of 750 g/ml. Western blot analysis.…”

“…Western blot analysis. Wound and cell culture lysates were prepared as described previously (17,20). Fifty micrograms of total protein from skin or cellular lysates was separated using SDS-gel electrophoresis.…”

p68 DEAD Box RNA Helicase Expression in Keratinocytes

“…Preparation of Protein Lysates and Cell Fractionation-Skin and cell culture samples were homogenized in lysis buffer (1% Triton X-100, 20 mM Tris/HCl, pH 8.0, 137 mM NaCl, 10% glycerol, 5 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 1% aprotinin, 15 g/ml leupeptin). The extracts were cleared by centrifugation (17,20). To obtain nuclear and cytoplasmic fractions of cultured cells and wound tissues, samples were homogenized in lysis buffer (1ϫ, without Triton).…”

Severely Impaired Insulin Signaling in Chronic Wounds of Diabetic ob/ob Mice

“…26,29 Fifty micrograms of total protein lysate was separated using sodium dodecyl sulfate (SDS)-gel electrophoresis, and specific proteins were detected using antisera directed against InsR␤, Glut-4 (Santa Cruz), PTP-1B, IRS-1, IRS-2 (Biomol, Hamburg, Germany), phospho-glycogen synthase kinase (GSK)-3␣/␤, phospho-glycogen synthase (GS), phospho-tyrosine (Cell Signaling, Frankfurt, Germany), or actin (Sigma, Deisenhofen, Germany).…”