Laminin and Fibronectin Treatment Leads to Generation of Dendritic Cells with Superior Endocytic Capacity

Copyright: 2010 Garcı´a-Nieto et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.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.Peer reviewedFinal Published versio

The Glycosylation Pattern of Common Allergens: The Recognition and Uptake of Der p 1 by Epithelial and Dendritic Cells Is Carbohydrate Dependent

Allergens are initiators of both innate and adaptive immune responses. They are recognised at the site of entry by epithelial and dendritic cells (DCs), both of which activate innate inflammatory circuits that can collectively induce Th2 immune responses. In an attempt to have a better understanding of the role of carbohydrates in the recognition and uptake of allergens by the innate immune system, we defined common glycosylation patterns in major allergens. This was done using labelled lectins and showed that allergens like Der p 1 (Dermatophagoides pteronyssinus group 1), Fel d 1 (Felis domisticus), Ara h 1 (Arachis hypogaea), Der p 2 (Dermatophagoides pteronyssinus group 2), Bla g 2 (Blattella germanica) and Can f 1 (Canis familiaris) are glycosylated and that the main dominant sugars on these allergens are 1–2, 1–3 and 1–6 mannose. These observations are in line with recent reports implicating the mannose receptor (MR) in allergen recognition and uptake by DCs and suggesting a major link between glycosylation and allergen recognition. We then looked at TSLP (Thymic Stromal Lymphopoietin) cytokine secretion by lung epithelia upon encountering natural Der p 1 allergen. TSLP is suggested to drive DC maturation in support of allergic hypersensitivity reactions. Our data showed an increase in TSLP secretion by lung epithelia upon stimulation with natural Der p 1 which was carbohydrate dependent. The deglycosylated preparation of Der p 1 exhibited minimal uptake by DCs compared to the natural and hyperglycosylated recombinant counterparts, with the latter being taken up more readily than the other preparations. Collectively, our data indicate that carbohydrate moieties on allergens play a vital role in their recognition by innate immune cells, implicating them in downstream deleterious Th2 cell activation and IgE production

Uniform cell colonization of porous 3-D scaffolds achieved using radial control of surface chemistry

Uniform cellular distribution is a prerequisite to forming tissue within porous scaffolds, but the seeding process often results in preferential adhesion of cells at the periphery. We develop a vapour phase coating strategy which is readily applicable to any porous solid to provide a uniform cellular distribution. Plasma polymerized allyl amine (ppAAm) is used to form a thin nitrogen-containing coating throughout porous three-dimensional (3-D) poly(d,l-lactic acid) scaffolds. Subsequent controlled deposition of a hydrocarbon plasma polymerized hexane (ppHex) allows control of the fibroblast penetration into these porous 3-D objects. In order to optimize the coating conditions, a planar pinhole model of plasma penetration into pores is developed to rapidly measure deposit penetration using picolitre water contact angle measurement. Sufficiently good control over the plasma deposition within the porous scaffold is achieved using this approach to superimpose a relatively cell-repellent ppHex coating at the scaffold periphery onto the ppAAm-coated core, with a chemical gradient between the two. This 3-D chemical gradient encourages 3T3 fibroblast cells to adhere homogeneously from the periphery to the centre, when balanced by the tortuousity of the pore structure, which cells experience when passing from the surrounding medium to the centre. © 2011 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved

Confocal images of the difference between recombinant and natural Der p 1 (0.5 µg/ml) uptake by the same immature DC at 37°C.

<p>The results suggest that the uptake of the recombinant preparation (A) is higher than that for natural Der p 1 (B) in the same DC. A. Green: rDer p 1 labelled with FITC, red: MR labelled with PE, blue: nucleus labelled with DAPI. B. Green: nDer p 1 labelled with Cy5, red: MR labelled with PE, blue: nucleus labelled with DAPI.</p

Binding of MR C-type lectin-like carbohydrate recognition domains 4–7 with different glycoforms of Der p 1, nDer p 1 and rDer p 1 (concentrations at 2 µg/ml).

<p>***<i>P</i> value<0.001.</p

The uptake of recombinant and natural preparations of Der p 1 (1 µg/ml) by immature DCs at 30 mins.

<p>A. Green: rDer p1 stained with FITC, red: MR stained with PE, blue: nucleus stained with DAPI. B. Green: nDer p 1 stained with Cy5, red: MR stained with PE, blue: nucleus stained with DAPI.</p

The co-localization of natural and recombinant Derp1 (0.5 µg/ml) with LAMP-2 detected at 10 mins.

<p>A. Green: rDer p 1 stained with FITC, red: LAMP-2 stained with PE, blue: nucleus stained with DAPI. B. Green: nDer p 1 stained with Cy5, red: LAMP-2 stained with PE, blue: nucleus stained with DAPI.</p

Commassie blue stained gel of natural Der p 1 before and after deglycosylation with periodate showing a slight decrease in the MW of deglycosylated Der p 1.

<p>Commassie blue stained gel of natural Der p 1 before and after deglycosylation with periodate showing a slight decrease in the MW of deglycosylated Der p 1.</p

Comparative analysis of cysteine protease allergens and non-allergens in terms of mannosylation.

<p>Allergens are strongly mannosylated and have stronger reaction with anti-mannose GNA compared to non-allergens. +++: strong reaction, ++: moderate reaction, +: mild reaction, −: no reaction.</p