Label-free molecular imaging of immunological synapses between dendritic and T cells by Raman micro-spectroscopy.

Confocal Raman micro-spectroscopy (CRMS) was used to measure spectral images of immunological synapse formation between dendritic and T cells without using molecular labels or other invasive procedures. The purpose-built inverted CRMS instrument integrated an environmental enclosure and a near-infrared laser to allow measurements on live cells maintained under physiological conditions. The integration of the wide-field fluorescence also enabled viability assays and direct comparison between Raman spectral images and gold-standard immuno-fluorescence images for specific molecules. Raman spectral images of nucleus and proteins were built by fuzzy c-mean clustering method. The Raman images were found to be in good correspondence with the immuno-fluorescence images of DNA and actin. These results indicate that actin is a main contributor to the Raman spectrum of the cytoplasm of dendritic and T cells. While for control cells the Raman spectral images of proteins indicated a more homogeneous distribution of proteins in the cytoplasm of dendritic cells, they indicated a higher accumulation of proteins at the immunological synapses when dendritic cells were pre-treated with laminin. These conclusions were also supported by confocal immuno-fluorescence imaging after cell fixation and labelling. This study demonstrates the potential of CRMS for label-free non-invasive imaging of junctions between live cells. Therefore, this technique may become a useful tool for studying cellular processes in live cells and where non-invasive molecular specific imaging is desirable, such as cell-cell interactions.

Laminin and fibronectin treatment leads to generation of dendritic cells with superior endocytic capacity.

BACKGROUND: Sampling the microenvironment at sites of microbial exposure by dendritic cells (DC) and their subsequent interaction with T cells in the paracortical area of lymph nodes are key events for initiating immune responses. Most of our knowledge of such events in human is based on in vitro studies performed in the absence of extracellular matrix (ECM) proteins. ECM in basement membranes and interstitial spaces of different tissues, including lymphoid organs, plays an important role in controlling specific cellular functions such as migration, intracellular signalling and differentiation. The aim of this study was, therefore, to investigate the impact of two abundant ECM components, fibronectin and laminin, on the phenotypical and functional properties of DC and how that might influence DC induced T-cell differentiation. METHODOLOGY/PRINCIPAL FINDINGS: Human monocyte derived DC were treated with laminin and fibronectin for up to 48 hours and their morphology and phenotype was analyzed using scanning electron microscopy, flow cytometry and real time PCR. The endocytic ability of DC was determined using flow cytometry. Furthermore, co-culture of DC and T cells were established and T cell proliferation and cytokine profile was measured using H(3)-thymidine incorporation and ELISA respectively. Finally, we assessed formation of DC-T cell conjugates using different cell trackers and flow cytometry. Our data show that in the presence of ECM, DC maintain a 'more immature' phenotype and express higher levels of key endocytic receptors, and as a result become significantly better endocytic cells, but still fully able to mature in response to stimulation as evidenced by their superior ability to induce antigen-specific T cell differentiation. CONCLUSION: These studies underline the importance of including ECM components in in vitro studies investigating DC biology and DC-T cell interaction. Within the context of antigen specific DC induced T cell proliferation, inclusion of ECM proteins could lead to development of more sensitive assays.

Internalization of a non-pathogenic mycobacteria by macropinocytosis in human alveolar epithelial A549 cells.

Mycobacterium smegmatis (MSM) a non-pathogenic mycobacterium is often employed as a tool to understand many aspects of the mycobacterial infections. However, its own biology and particularly its mechanism of entry into non-phagocytic cells are not well known. Previously, we demonstrated that Mycobacterium tuberculosis (MTB) invades epithelial cells by macropinocytosis. In the present study, we investigated whether MSM also invades human epithelial type II pneumocytes (A549) by macropinocytosis. Infection of A549 cells with MSM elicited actin filaments redistribution, lamellipodia formation and increased fluid phase uptake, suggesting macropinocytosis. Furthermore, macropinocytosis inhibitors like cytochalasin D and amiloride caused inhibition of fluid phase and bacterial uptake. We can conclude that MSM, like MTB, takes advantage of macropinocytosis for entry into epithelial cells, however, unlike MTB, internalized MSM are killed by host cells. These findings suggest that induction of macropinocytosis and cell invasion is not an exclusive feature of pathogenic organisms.