Induction of high expression of CCR7 and high production of IL-12 in human monocyte-derived dendritic cells by a new bacterial component: LCOS 1013.

Dendritic cells (DCs) are the most potent antigen presenting cells of the immune system as they can act as initiators, stimulators and regulators of the immune response. Human DCs are most commonly generated for clinical use by in vitro differentiation of monocytes with exogenous cytokines. Here, we investigate the effect of LCOS 1013 on the production of mature Mo-DCs. LCOS 1013 is a new bacterial component from walls of gram(+)Klebsellia pneumoniae bacteria that contain some OmpA glycoproteins. Purified peripheral blood monocytes were cultured for 6 days with IL-4 and GM-CSF in order to obtain immature dendritic cells (Im-MoDCs). On day six, Im-MoDCs were matured with either LCOS 1013, TNF alpha, LPS or CD40-Ligand. LCOS 1013 matured Mo-DCs (LCO-DCs) showed a higher expression of DC-LAMP, CD80, CD83, CD54 and CD40 than TNF alpha, LPS and CD40L matured Mo-DCs. Interestingly, LCO-DCs exhibited high expression of full competent CCR7 and high secretion of IL-12 during their maturation. Functionally, LCO-DCs have equivalent potency to trigger mixed leukocyte reaction and antigen-specific reaction and polarize immune response towards Th1 way. Moreover, we found that LCOS 1013 activates DCs through TLR2. LCOS 1013 represents an attractive therapeutic maturation agent of DCs allowing the production of Mo-DCs with high capacity to migrate and to induced Th1 immune responses.

Identification and quantification of fetal red blood cells in maternal blood by a dual-color flow cytometric method: evaluation of the Fetal Cell Count kit.

BACKGROUND: As an alternative to the cumbersome Kleihauer-Betke test (KBT), flow cytometry represents a powerful method for the identification and quantification of fetal red blood cells (RBCs) in maternal circulation. STUDY DESIGN AND METHODS: The aim of this study was to evaluate the Fetal Cell Count kit (IQ Products), an innovative flow cytometric method, based on the combination of antibodies directed, respectively, against fetal hemoglobin (HbF) and carbonic anhydrase (CA), a marker expressed after birth, to discriminate fetal RBCs from adult F cells containing HbF. The investigation was performed by two French laboratories that compared the data obtained by flow cytometry and KBT in 455 pregnant or just-delivered women as well as in 124 artificial mixtures containing from 0.01 to 5.00 percent cord cells. RESULTS: The FL1/FL2 histogram allowed distinction between fetal RBCs (HbF+, CA-), F cells (HbF+, CA+), and adult RBCs (HbF-, CA+). The limits of detection and quantification were determined at 0.03 and 0.10 percent or 0.02 and 0.05 percent when analyzing 100,000 or 200,000 events, respectively. Linearity was demonstrated between 0.01 and 5.00 percent fetal cells in the mixtures (r = 0.95, p < 0.01). A good correlation between fluorescence-activated cell sorting (FACS) and KBT results was obtained with artificial mixtures (r = 0.94, p < 0.01). From the 405 Kleihauer-negative samples, none were identified as positive by FACS. Among the 50 Kleihauer-positive samples, 6 were shown not to contain fetal cells but F cells by FACS. CONCLUSION: With this new dual-color flow cytometric method, accurate evaluation of fetomaternal hemorrhage was achieved even in the face of HbF of maternal origin.

Toll-like receptor 3 (TLR3): a new marker of canine monocytes-derived dendritic cells (cMo-DC).

Toll-like receptors (TLRs) are a family of functionally important receptors for recognition of pathogen-associated molecular pattern (PAMP) since they trigger the pro-inflammatory response and upregulation of costimulatory molecules, linking the rapid innate response to adaptative immunity. In human leukocytes, TLR3 has been found to be specifically expressed in dendritic cells (DC). This study examined the expression of TLR3 in canine monocytes-derived DC (cMo-DC) and PBMC using three new anti-TLR3 mAbs (619F7, 722E2 and 713E4 clones). The non-adherent cMo-DC generated after culture in canine IL-4 plus canine GM-CSF were labelled with the three anti-TLR3 clones by flow cytometry, with a strong expression shown for 619F7 and 722E2 clones. By contrast, TLR3 expression was low to moderate in canine monocytes and lymphocytes. These results were confirmed by Western blot using 619F7 and 722E2 clones and several polypeptide bands were observed, suggesting a possible cleavage of TLR3 molecule or different glycosylation states. In addition, TLR3 was detectable in immunocytochemistry by using 722E2 clone. In conclusion, this first approach to study canine TLR3 protein expression shows that three anti-TLR3 clones detect canine TLR3 and can be used to better characterize canine DC and the immune system of dogs.

Rabbit antithymocyte globulin inhibits monocyte-derived dendritic cells maturation in vitro and polarizes monocyte-derived dendritic cells towards tolerogenic dendritic cells expressing indoleamine 2,3-dioxygenase.

BACKGROUND: Rabbit antithymocyte globulin (rATG) is a polyclonal mixture of immunoglobulin (Ig) G. It is used to prevent graft rejection and also graft versus host disease after transplantation. Its effect on lymphocyte function has been widely studied. Dendritic cells are central actors of the immune system. As antigen presenting cells, they are able to initiate, stimulate, and modulate immune responses. METHODS: In this study, we investigated rATG effects on in vitro differentiation and maturation of monocyte-derived dendritic cells (Mo-DCs). RESULTS: rATG inhibited maturation of immature Mo-DCs and allowed the generation of dendritic cells expressing ILT-3, CD123, CCR6 but not CCR7 and producing Indoleamine 2,3-dioxygenase mRNA as well as interferon-alpha. CONCLUSION: rATG polarizes in vitro Mo-DCs towards tolerogenic dendritic cells.

CD86 molecule is a specific marker for canine monocyte-derived dendritic cells.

In this study, canine monocyte-derived dendritic cells (cMo-DC) were produced in presence of canine GM-CSF (cGM-CSF) and canine IL-4 (cIL-4), and they were characterized by their dendritic morphology, MLR functionality and phenotype. We noticed that cMo-DC were labelled with three anti-human CD86 (FUN-1, BU63 and IT2.2 clones), whereas resting and activated lymphocytes or monocytes were not stained. CD86 expression was induced by cIL-4 and was up-regulated during the differentiation of the cMo-DC, with a maximum at day 7. Furthermore, cMo-DC were very potent even in low numbers as stimulator cells in allogeneic MLR, and BU63 mAb was able to completely block the cMo-DC-induced proliferation in MLR. We also observed that cMo-DC highly expressed MHC Class II and CD32, but we failed to determine their maturation state since the lack of commercially available canine markers. Moreover, cMo-DC contained cytoplasmic periodic microstructures, potentially new ultrastructural markers of canine DC recently described. In conclusion, this work demonstrates that the CD86 costimulatory marker is now usable for a better characterization of in vitro canine DC.

Integrin alpha1beta1 mediates collagen induction of MMP-13 expression in MC615 chondrocytes.

During endochondral ossification, type I collagen is synthesized by osteoblasts together with some hypertrophic chondrocytes. Type I collagen has also been reported to be progressively synthesized in degenerative joints. Because Matrix Metalloproteinase-13 (MMP-13) plays an active role in remodeling cartilage in fetal development and osteoarthritic cartilage, we investigated whether type I collagen could activate MMP-13 expression in chondrocytes. We used a well-established chondrocytic cell line (MC615) and we found that MMP-13 expression was induced in MC615 cells cultured in type I collagen gel. We also found that alpha1beta1 integrin, a major collagen receptor, was expressed by MC615 cells and we further assessed the role of alpha1beta1 integrin in conducting MMP-13 expression. Induction of MMP-13 expression by collagen was potently and synergistically inhibited by blocking antibodies against alpha1 and beta1 integrin subunits, indicating that alpha1beta1 integrin mediates the MMP-13-inducing cellular signal generated by three-dimensional type I collagen. We also determined that activities of tyrosine kinase and ERK and JNK MAP kinases were required for this collagen-induced MMP-13 expression. Interestingly, bone morphogenetic protein (BMP)-2 opposed this induction, an effect that may be related to a role of BMP-2 in the maintenance of cartilage matrix.

Evaluation of elutriation and magnetic microbead purification of canine monocytes.

An elutriation technique was developed to obtain large quantities of pure canine monocytes. Firstly, peripheral blood mononuclear cells (PBMC) were isolated from whole blood by Ficoll gradient. Then, the PBMC were separated by an elutriation procedure. We demonstrated that these techniques allow the isolation of canine peripheral blood monocytes with a purity of 64% +/- 7.9 when labelled with anti-CD14 antibody. This purity increased to 83% +/- 2.2 after separation by magnetic anti-CD14 microbeads. The cell viability was more than 95% and apoptotic cells were less than 10%. The monocytes purified by these methods were functionally active in a mixed leukocyte reaction (MLR). A lymphocyte fraction was obtained directly only by elutriation with an average of 79.9% +/- 10.7 of CD5+, 7.9% +/- 3.5 of CD21+ and 1.78% +/- 2.53 of CD14+. Our results indicate that this elutriation procedure is a safe method to purify monocytes as well as lymphocytes, useful in MLR.

Normal human keratinocytes bind to the alpha3LG4/5 domain of unprocessed laminin-5 through the receptor syndecan-1.

Basal keratinocytes of the epidermis adhere to their underlying basement membrane through a specific interaction with laminin-5, which is composed by the association of alpha3, beta3, and gamma2 chains. Laminin-5 has the ability to induce either stable cell adhesion or migration depending on specific processing of different parts of the molecule. One event results in the cleavage of the carboxyl-terminal globular domains 4 and 5 (LG4/5) of the alpha3 chain. In this study, we recombinantly expressed the human alpha3LG4/5 fragment in mammalian cells, and we show that this fragment induces adhesion of normal human keratinocytes and fibrosarcoma-derived HT1080 cells in a heparan- and chondroitin sulfate-dependent manner. Immunoprecipitation experiments with Na2 35SO4-labeled keratinocyte and HT1080 cell lysates as well as immunoblotting experiments revealed that the major proteoglycan receptor for the alpha3LG4/5 fragment is syndecan-1. Syndecan-4 from keratinocytes also bound to alpha3LG4/5. Furthermore we could show for the first time that unprocessed laminin-5 specifically binds syndecan-1, while processed laminin-5 does not. These results demonstrate that the LG4/5 modules within unprocessed laminin-5 permit its cell binding activity through heparan and chondroitin sulfate chains of syndecan-1 and reinforce previous data suggesting specific properties for the precursor molecule.

Keratinocyte motility induced by TGF-beta1 is accompanied by dramatic changes in cellular interactions with laminin 5.

Transforming growth factor-beta1 (TGF-beta1) has the ability to induce epithelial cell migration while stopping proliferation. In this study, we show that, concomitant to promoting migration of normal human keratinocytes in vitro, TGF-beta1 induced a marked decrease in their adhesion capacity to processed alpha3-containing laminin 5-coated surfaces. Indeed, the expression levels of alpha3 and alpha6 integrin subunit mRNA and protein, as well as the cell surface alpha3beta1 and alpha6beta4 integrins, were down-regulated. Recent studies showed that keratinocytes over express and deposit laminin 5 during migration and we have shown that laminin 5 found in the matrix of TGF-beta1 induced migrating keratinocytes is present in its unprocessed form [Décline and Rousselle, 2001: J. Cell Sci. 114:811-823]. We show here that TGF-beta1 treatment of the cells promoted a significant increase in their adhesion to the alpha3 chain carboxy-terminal LG4/5 subdomain and that this interaction is likely to be mediated by a heparan sulfate proteoglycan type of receptor. Our results indicate that alpha6beta4 and alpha3beta1 integrin interactions with laminin 5 are diminished during migration while a specific interaction occurs between an additional cellular receptor and the alpha3 LG4/5 module present on unprocessed laminin 5.

Generation of monocyte-derived dendritic cells in patients with hereditary hemochromatosis.

Hereditary hemochromatosis (HH) is a common genetic disease with autosomal recessive transmission and is characterized by a dysregulation of iron metabolism, leading to serum iron overload and its progressive accumulation in most body tissues. The effects of HH on the immune system include altered lymphocytosis and functions of monocytes. Moreover, monocytes can differentiate into dendritic cells (DCs), which play crucial roles in the immune response (capture, processing, and presentation of antigen to effector T cells) and this process was shown to be impaired in several pathologies. The aim of this study was to determine whether the monocytes from HH patients still displayed the ability to differentiate into DCs. To that purpose, purified monocytes from healthy donors and HH patients were cultured in the appropriate medium. The results showed no phenotypic and functional differences, at both the immature and the mature stages. Furthermore, our work reports altered lymphocytosis with expanded CD8+CD28- T cell subset. These monocyte-derived DCs could therefore be a solid vector for DC-based immunotherapy and a powerful tool for investigating the immune regulatory loops and especially the biological relevance of the expanded CD8+CD28- T cells since this population has also been described as suppressor T cells.