LIMLE, a new molecule over-expressed following activation, is involved in the stimulatory properties of dendritic cells

Abstract

Dendritic cells are sentinels of the immune system distributed throughout the body, that following danger signals will migrate to secondary lymphoid organs to induce effector T cell responses. We have identified, in a rodent model of graft rejection, a new molecule expressed by dendritic cells that we have named LIMLE (RGD1310371). To characterize this new molecule, we analyzed its regulation of expression and its function. We observed that LIMLE mRNAs were rapidly and strongly up regulated in dendritic cells following inflammatory stimulation. We demonstrated that LIMLE inhibition does not alter dendritic cell maturation or cytokine production following Toll-like-receptor stimulation. However, it reduces their ability to stimulate effector T cells in a mixed leukocyte reaction or T cell receptor transgenic system. Interestingly, we observed that LIMLE protein localized with actin at some areas under the plasma membrane. Moreover, LIMLE is highly expressed in testis, trachea, lung and ciliated cells and it has been shown that cilia formation bears similarities to formation of the immunological synapse which is required for the T cell activation by dendritic cells. Taken together, these data suggest a role for LIMLE in specialized structures of the cytoskeleton that are important for dynamic cellular events such as immune synapse formation. In the future, LIMLE may represent a new target to reduce the capacity of dendritic cells to stimulate T cells and to regulate an immune response.

Figure 1. LIMLE mRNA expression in transplantation models, cell subtypes and organs.

LIMLE mRNA expression in (A) cardiac grafts and PBMC of rat syngenic recipients (Syng), allograft recipients developing acute (AR) or chronic rejection (CR), or allograft tolerant recipients (Tol) at day 5 or 100 after transplantation (n = 5, *p<0.05 **p<0.01 ***p<0.001), (B) Splenic DCs, alveolar macrophage lineage cells NR8383 (Mac), B cells, T cells and EC, stimulated (stim) or not (NS) with LPS, IFNγ, CpG, anti-CD3/CD28 or IFNγ respectively (for 12 hours) (n = 3), (C) rat BMDCs stimulated or not (NS) with LPS, IFNγ, CpG, Poly(I:C) or IL-10 for 6, 12, 24 or 48 hours (n = 3, *p<0.05), (D) Human DCs stimulated or not (NS) with LPS for 12 hours (n = 3, **p<0.01) (E) mRNA expression of LIMLE in different organs from naive rats as indicated (n = 5). (A–E) Quantitative RT-PCR results were expressed in Arbitrary Units (AU) of LIMLE/HPRT transcript ratio ± SEM. Statistical evaluation was performed using Student's t test for unpaired data, and results were considered significant if p values were <0.05.

Figure 2. The role of LIMLE in the stimulatory properties of BMDCs.

(A) a) LIMLE mRNA expression in rat BMDCs transfected with control or one of two LIMLE specific RNAi. Quantitative RT-PCR results were expressed in Arbitrary Unit (AU) of LIMLE/HPRT transcript ratio ± SEM (n = 3 *p<0.05 ***p<0.001); b) V5 tagged LIMLE protein expression in eukaryote cells transfected with a plasmid encoding rat LIMLE and V5 and either a control or one of two LIMLE specific RNAi. Data were expressed in Arbitrary Units (AU) of % of V5⁺ cells assessed by flow cytometry compared to control RNAi  = 100 (n = 6, **p<0.01) (B) Proliferation of allogeneic T cells stimulated with control or one of two LIMLE specific RNAi transfected BMDCs (MLR) for 4 days as follow a) ³H incorporation (cpm) for bulk T cells, b) CFSE loss from CD4⁺CD25^neg and c) CFSE loss from CD4⁺CD25^high T cells (n≥3, *p<0.05). (C) IFNγ, IL-6 and IL-12 quantification by ELISA of MLR supernatants (n = 3, *p<0.05). (D) Representative histograms (flow cytometry) of mouse BMDC transfection efficiency (with red fluorescent control RNAi (plain line) or untransfected control (dotted line)). (E) LIMLE mRNA expression in mouse BMDCs transfected with control or one of two LIMLE specific RNAi. Quantitative RT-PCR results were expressed in Arbitrary Units (AU) of LIMLE/GAPDH transcript ratio ± SEM (n = 3, *p<0.05 ***p<0.001). (F) Proliferation of OT-I CD8⁺ T cells stimulated for 4 days with OVA_257–264 peptide (0.1 ng/ml) (n = 3) or OVA protein (500 μg/ml) (n = 4) loaded mouse BMDCs (transfected with control or one of two LIMLE specific RNAi). Data were expressed in AU of CFSE dilution compared to control = 100 (*p<0.05 **p<0.01 ***p<0.001). Statistical evaluation was performed using the Student's t test for unpaired data, and results were considered significant if p values were <0.05.

Figure 3. Cellular localization of LIMLE protein.

Representative pictures of immuno-fluorescence staining of (A and B) COS or (C) BMDCs transfected with a plasmid encoding full length rat LIMLE protein and containing the V5 tag (red) with DAPI (blue) and and phalloidin (polymerized actin) (green). Original magnification ×1200 (Plan Apo N.A: 1.4 zoom 2.). (Bi,ii) Representative histograms of fluorescence intensity (AU) in COS cells detected in pixels with the indicated box, graphed with the linescan function of Metamorph Image Processing Software. (Ci) Representative picture of LIMLE overlapping with actin in DCs, processed with Metamorph Image Processing Software. Pictures were representative of three independent experiments.