Abstract
Abstract: :
Purpose: T lymphocyte migration in lymphoid tissue has been well documented but in vivo visualization of T cell movement at a site of inflammation is relatively unstudied. We have characterized T cell movement in an enhanced iritis model using a minor modification of a previously described model of ovalbumin (OVA)–induced uveitis. Methods: OVA–specific T cells isolated from spleens of DO11.10 transgenic mice were cultured in vitro with OVA323–329 peptide, labeled with CMTMR fluorescent dye, and injected IV into four naïve BALB/c mice (approximately 2x107 cells/animal). After 2–3 days, these mice received a subconjunctival injection of 150 mg chicken ovalbumin (Sigma, grade V) in PBS and one day later were challenged in the anterior chamber (AC) with the same antigen. Twenty–four hours following AC challenge, fluorescent T cells were imaged for 30 min in irises of anesthetized mice by intravital fluorescence time–lapse videomicroscopy. Computerized image stabilization was used to compensate for eye movement (MediaCybernetics) and individual cells were tracked using NIH ImageJ v1.30. Because some background iris movement remained after stabilization, individual T cell motion was analyzed relative to a nearby non–migrating cell or blood vessel junction. Cell speeds were determined for 20–second increments. Results: The addition of a subconjunctival injection of OVA 24 h prior to the AC injection increases the resulting iritis substantially such that videomicroscopy of the infiltrating DO11.10 cells is facilitated. In contrast to what we have reported for neutrophils in the iris stroma, the migratory behavior of labeled T cells varied considerably within our recording interval. About 50% of the cells did not move at all (4.4±2.9 µm/min (mean±SD) comparable to background movement of the iris itself) and about one in six moved slightly (5.0±3.9 µm/min). Only a few cells were classified as highly mobile with the fastest three cells having an average speed of 9.4±5.6 µm/min. Cell path tracings and mean displacement graphs revealed that some migration was directed, some was restricted, and much appeared random. A given cell might display predominantly one motion type or might switch between 2 or 3 types. Conclusions: This is the first report to use time–lapse imagery to characterize T cell migration in uveitis. T cell migration patterns display substantial diversity. This model should be readily adapted to study T cell migration with respect to the position of antigen–presenting cells and blood vessels.
Keywords: uveitis-clinical/animal model • microscopy: light/fluorescence/immunohistochemistry • imaging/image analysis: non-clinical