Characterizing extravascular neutrophil migration in vivo in the iris.

Extravascular neutrophil migration is poorly characterized in vivo. To test the hypothesis that this migration is a non-random process, we used videomicroscopy to monitor neutrophils in irises of living mice with endotoxin-induced uveitis (EIU). Paths of individual cells were analyzed. Nearly all of these cells were moving in divergent directions, and mean displacement plots indicated that the predominant movement was random. The paths of some cells were fit to bivariate autoregressive integrated moving average models that revealed at least two modes of movement: random search and linear trend. Cell speed was significantly reduced by the actin inhibitor, cytochalasin D. The pattern of migration for neutrophils is in marked contrast to what we previously described for antigen-presenting cells in the iris, but somewhat resembles recent descriptions for T cells within a lymph node. Characterization of extravascular migration of neutrophils has important implications for understanding infection and immunity.

Matrix-coated transwell-cultured TM4 sertoli cell testosterone-regulated gene expression mimics in vivo expression.

In vitro culture systems are needed to mimic in vivo epithelial cell environments for identifying cell signaling, gene expressions, and molecular mechanisms. One such system is matrix-coated transwell cultures. However, no data exist on culturing Sertoli cells in this manner with respect to testosterone-regulated gene expression. Because the TM4 mouse Sertoli-like cell line expresses androgen receptor, our objective was to determine if testosterone treatment added to the bottom chamber of a matrix-coated transwell system induces some gene expressions found in Sertoli cells in vivo. After serum starvation, transwell-cultured TM4 cells were treated with testosterone or left untreated for 24 h. Microarray analyses initially identified differentially expressed genes either induced or repressed by testosterone treatment. By Northern blot analyses, Pem mRNA, a frequently used marker of Sertoli cell testosterone responsiveness, was induced. Proteins of the transcripts induced by testosterone in the in vitro system were immunolocalized to Sertoli cells in testosterone-dependent stages of spermatogenesis in mouse testes. By immunohistochemistry analyses of sectioned mouse testes, gene expression induced by testosterone in transwell-cultured TM4 cells, profilin as well as secreted protein acidic and rich in cysteines (SPARC) are localized to Sertoli cells in testosterone-dependent stages of spermatogenesis. Findings include localizations of SPARC and profilin, as well as an apparent germ cell communication required for translation of Pem mRNA in Sertoli cells. Taken together, results of these studies suggest that this TM4 transwell-culture system could be used to study these testosterone-regulated Sertoli gene expressions in vitro.

Overexpression of carcinoma and embryonic cytotrophoblast cell-specific Mig-7 induces invasion and vessel-like structure formation.

Molecular requirements for carcinoma cell interactions with the microenvironment are critical for disease progression but are poorly understood. Integrin alpha v beta 5, which senses the extracellular matrix, is important for carcinoma cell dissemination in vivo. alpha v beta 5 signaling induces Mig-7, a novel human gene product that is apparently carcinoma-specific. We hypothesized that Mig-7 expression facilitates tumor cell dissemination by increasing invasion and vasculogenic mimicry. Results show that embryonic cytotrophoblasts up-regulated Mig-7 expression before they acquired an invasive phenotype capable of pseudovasculogenesis. Mig-7 protein primarily co-localized with vasculogenic mimicry markers factor VIII-associated antigen, vascular endothelial-cadherin, and laminin 5 gamma 2 chain domain III fragment in lymph node metastases. Overexpression of Mig-7 increased gamma 2 chain domain III fragments known to contain epidermal growth factor (EGF)-like repeats that can activate EGF receptor. Interestingly, EGF also induced Mig-7 expression. Carcinoma cell adhesion to laminins was significantly reduced by Mig-7 expression. Remarkably, in two-dimensional and three-dimensional Matrigel cultures, Mig-7 expression caused invasion and vessel-like structures. Melanoma cells, which were previously characterized to invade aggressively and to undergo vasculogenic mimicry, expressed Mig-7. Taken together, these data suggest that Mig-7 expression allows cells to sense their environment, to invade, and to form vessel-like structures through a novel relationship with laminin 5 gamma 2 chain domain III fragments.

Antigen-specific accumulation of naïve, memory and effector CD4 T cells during anterior uveitis monitored by intravital microscopy.

Uveitis is an immune-mediated ocular disease and a leading cause of blindness. We characterized a novel model of uveitis with intravital microscopy. Transfer of ovalbumin-specific T cells from DO11.10 spleen to BALB/c recipients and subsequent challenge with ovalbumin in the anterior chamber of the eye resulted in anterior uveitis. Antigen-specificity was verified by injection of irrelevant antigen and transfer of T cells with a different specificity. Subsets of CD4 T cells, including naive (DO11.10 RAG(-/-)) and in vitro-activated Th2 effector CD4 T cells, infiltrated anterior segment tissues early in the inflammation. Memory-like CD44(high) CD4 T cells from unprimed transgenic mice and in vitro-activated Th1 effector CD4 T cells accumulated to larger numbers than naive or Th2 effector cells at 48 and 72 h. Of these, the alpha(2)-integrin+CD4 unprimed T cells entered the eye more efficiently, and antibody to alpha(2)-integrin markedly inhibited the inflammatory response. Intravital microscopy revealed the early arrival and antigen-specific accumulation of CD4 T cells in inflamed tissue and should be helpful in understanding T cell migration to other organs.

APCs in the anterior uveal tract do not migrate to draining lymph nodes.

The migration of APCs from sites of infection and their maturation are critical elements in the generation of immune responses. However, the paths by which intraocular Ags migrate to draining lymph nodes are not known because the eye has limited lymphatic vessels. To date, only dendritic cells from the cornea and conjunctiva have been shown to emigrate. We demonstrate that phagocytic APCs in the anterior uveal tissues of the murine eye that ingest fluorescent latex beads do not migrate to regional lymph nodes. The beads are ingested in the uveal tract by cells expressing MHC class II, CD11c, or F4/80. Using intravital time-lapse videomicroscopy to monitor iris APC migration after anterior chamber injection of fluorescent Ag, fluorescently labeled APCs fail to move at multiple observation times, even in the presence of Ag and LPS. Whereas an as yet unidentified ocular nonphagocytic APC subset might migrate from the anterior uveal tissues, it is more probable that immune responses in the draining lymph nodes are engendered by soluble Ag escaping the eye through interstitial spaces. The inability of anterior uveal tissue APCs to migrate to lymph nodes may contribute to deviant immune responses that dominate after Ags are introduced into the anterior chamber.