Donna Fluitsma

Langerin is a natural barrier to HIV-1 transmission by Langerhans cells.

Human immunodeficiency virus-1 (HIV-1) is primarily transmitted sexually. Dendritic cells (DCs) in the subepithelium transmit HIV-1 to T cells through the C-type lectin DC-specific intercellular adhesion molecule (ICAM)-3-grabbing nonintegrin (DC-SIGN). However, the epithelial Langerhans cells (LCs) are the first DC subset to encounter HIV-1. It has generally been assumed that LCs mediate the transmission of HIV-1 to T cells through the C-type lectin Langerin, similarly to transmission by DC-SIGN on dendritic cells (DCs). Here we show that in stark contrast to DC-SIGN, Langerin prevents HIV-1 transmission by LCs. HIV-1 captured by Langerin was internalized into Birbeck granules and degraded. Langerin inhibited LC infection and this mechanism kept LCs refractory to HIV-1 transmission; inhibition of Langerin allowed LC infection and subsequent HIV-1 transmission. Notably, LCs also inhibited T-cell infection by viral clearance through Langerin. Thus Langerin is a natural barrier to HIV-1 infection, and strategies to combat infection must enhance, preserve or, at the very least, not interfere with Langerin expression and function.

C-type lectin Langerin is a beta-glucan receptor on human Langerhans cells that recognizes opportunistic and pathogenic fungi.

Langerhans cells (LCs) lining the stratified epithelia and mucosal tissues are the first antigen presenting cells to encounter invading pathogens, such as viruses, bacteria and fungi. Fungal infections form a health threat especially in immuno-compromised individuals. LCs express C-type lectin Langerin that has specificity for mannose, fucose and GlcNAc structures. Little is known about the role of human Langerin in fungal infections. Our data show that Langerin interacts with both mannan and β-glucan structures, common cell-wall carbohydrate structures of fungi. We have screened a large panel of fungi for recognition by human Langerin and, strikingly, we observed strong binding of Langerin to a variety of Candida and Saccharomyces species and Malassezia furfur, but very weak binding was observed to Cryptococcus gattii and Cryptococcus neoformans. Notably, Langerin is the primary fungal receptor on LCs, since the interaction of LCs with the different fungi was blocked by antibodies against Langerin. Langerin recognizes both mannose and β-glucans present on fungal cell walls and our data demonstrate that Langerin is the major fungal pathogen receptor on human LCs that recognizes pathogenic and commensal fungi. Together these data may provide more insight in the role of LCs in fungal infections.

Human Langerhans cells capture measles virus through Langerin and present viral antigens to CD4+ T cells but are incapable of cross-presentation

Langerhans cells (LCs) are a subset of DCs that reside in the upper respiratory tract and are ideally suited to sense respiratory virus infections. Measles virus (MV) is a highly infectious lymphotropic and myelotropic virus that enters the host via the respiratory tract. Here, we show that human primary LCs are capable of capturing MV through the C‐type lectin Langerin. Both immature and mature LCs presented MV‐derived antigens in the context of HLA class II to MV‐specific CD4+ T cells. Immature LCs were not susceptible to productive infection by MV and did not present endogenous viral antigens in the context of HLA class I. In contrast, mature LCs could be infected by MV and presented de novo synthesized viral antigens to MV‐specific CD8+ T cells. Notably, neither immature nor mature LCs were able to cross‐present exogenous UV‐inactivated MV or MV‐infected apoptotic cells. The lack of direct infection of immature LCs, and the inability of both immature and mature LCs to cross‐present MV antigens, suggest that human LCs may not be directly involved in priming MV‐specific CD8+ T cells. Immune activation of LCs seems a prerequisite for MV infection of LCs and subsequent CD8+ T‐cell priming via the endogenous antigen presentation pathway.

A CD34+ human cell line model of myeloid dendritic cell differentiation: evidence for a CD14+CD11b+ Langerhans cell precursor

The study of early events in dendritic cell (DC) differentiation is hampered by the lack of homogeneous primary cell systems that allow the study of cytokine‐driven, transitional DC differentiation steps. The CD34+ acute myeloid leukemia cell line MUTZ‐3 displays a unique ability to differentiate into interstitial DC (IDC) and Langerhans cells (LC) in a cytokine‐dependent manner. Phenotypic characterization revealed MUTZ‐3 to consist of three distinct subpopulations. Small CD34+CD14−CD11b− progenitors constitute the proliferative compartment of the cell line with the ability to differentiate through a CD34−CD14−CD11b+ stage to ultimately give rise to a morphologically large, nonproliferating CD14+CD11bhi progeny. These CD14+CD11bhi cells were identified as common, immediate myeloid DC precursors with the ability to differentiate into LC and IDC, exhibiting characteristic and mutually exclusive expression of Langerin and DC‐specific ICAM‐grabbing nonintegrin, respectively. The identity of the MUTZ‐3‐derived LC subset was confirmed further by the presence of Birbeck granules. We conclude that the MUTZ‐3 cell line provides a ready and continuous supply of common myeloid precursors, which should facilitate further study of the ontogeny of myeloid DC lineages.

Blocking α2 integrins on rat CC531s colon carcinoma cells prevents operation‐induced augmentation of liver metastases outgrowth

Currently, an operation is the only curative option for patients with colorectal cancer. Unfortunately, many patients will develop liver metastases even after successful resection of the primary tumor. Removal of primary colorectal carcinoma may paradoxically increase the risk of metastases development, because accumulating evidence suggests that surgical trauma can stimulate tumor growth. In the present study, we investigated the effects of abdominal trauma on liver metastases development. Surgical trauma dramatically increased adhesion of tumor cells in the liver, leading to enhanced outgrowth of metastases. Endothelial stress was observed rapidly after an operation, suggesting that abdominal trauma resulted in impairment of blood vessel integrity. Tumor cells preferentially adhered to extracellular matrix (ECM). Furthermore, preincubation of tumor cells with anti‐α2 integrin antibodies completely reverted operation‐induced augmentation of CC531s adhesion and liver metastases outgrowth. As such, we postulate that blood vessel integrity in the liver is compromised after abdominal trauma, resulting in enhanced ECM exposure, which enables tumor cell adhesion and metastases outgrowth. Conclusion: Perioperative treatments that either aim to reduce endothelial stress or block the interaction between tumor cells and ECM represent promising new therapeutic strategies for the prevention of liver metastases development after resection of the primary tumor. (HEPATOLOGY 2007.)

Targeting FcαRI on Polymorphonuclear Cells Induces Tumor Cell Killing through Autophagy

Neutrophils are the most abundant circulating FcR-expressing WBCs with potent cytotoxic ability. Currently, they are recognized as promising effector cells for Ab-mediated immunotherapy of cancer, because their capacity to kill tumor cells is greatly enhanced by tumor Ag-specific mAbs. The FcαRI represents the most potent FcR on neutrophils for induction of Ab-mediated tumor cell killing. However, the mechanisms of cell death that are induced are poorly understood. Because these mechanisms can be used for modulation of anticancer treatment, we investigated the tumor cell death induced by neutrophil-mediated Ab-dependent killing via FcαRI. Human mammary carcinoma cells were efficiently killed when incubated with human neutrophils and tumor-specific FcαRI bispecific or IgA Abs. Interestingly, we observed characteristics of autophagy such as autophagic structures by electron microscopy and LC3B+ autophagosomes in different human epithelial carcinoma cells, which resulted in tumor cell death. To a lesser extent, necrotic features, such as cellular membrane breakdown and spillage of intracellular content, were found. By contrast, apoptotic features including fragmented nuclei, Annexin V-positivity, and presence of cleaved caspase-3 were not observed. These findings indicate that neutrophils mainly facilitate autophagy to induce tumor cell death rather than the more commonly recognized apoptotic cell death mechanisms induced by NK cells or cytotoxic T cells. This knowledge not only reveals the type of tumor cell death induced in neutrophil-mediated, Ab-dependent cellular cytotoxicity, but importantly opens up additional perspectives for modulation of anticancer therapy in, for example, apoptosis-resistant tumor cells.

Caveolin-1 mediated uptake via langerin restricts HIV-1 infection in human Langerhans cells

BackgroundHuman Langerhans cells (LCs) reside in foreskin and vaginal mucosa and are the first immune cells to interact with HIV-1 during sexual transmission. LCs capture HIV-1 through the C-type lectin receptor langerin, which routes the virus into Birbeck granules (BGs), thereby preventing HIV-1 infection. BGs are langerin-positive organelles exclusively present in LCs, however, their origin and function are unknown.ResultsHere, we not only show that langerin and caveolin-1 co-localize at the cell membrane and in vesicles but also that BGs are langerin/caveolin-1-positive vesicles are linked to the lysosomal degradation pathway in LCs. Moreover, inhibition of caveolar endocytosis in primary LCs abrogated HIV-1 sequestering into langerin+ caveolar structures. Notably, both inhibition of caveolar uptake and silencing of caveolar structure protein caveolin-1 resulted in increased HIV-1 integration and subsequent infection. In contrast, inhibition of clathrin-mediated endocytosis did not affect HIV-1 integration, even though HIV-1 uptake was decreased, suggesting that clathrin-mediated endocytosis is not involved in HIV-1 restriction in LCs.ConclusionsThus, our data strongly indicate that BGs belong to the caveolar endocytosis pathway and that caveolin-1 mediated HIV-1 uptake is an intrinsic restriction mechanism present in human LCs that prevents HIV-1 infection. Harnessing this particular internalization pathway has the potential to facilitate strategies to combat HIV-1 transmission.

Mutz-3-derived Langerhans cells are a model to study HIV-1 transmission and potential inhibitors

Sexual transmission is the primary route of HIV‐1 infection, and DC subsets are thought to be involved in viral dissemination to T cells. In the genital mucosa, two main subsets of DCs are present: epithelial LCs capture and degrade HIV‐1 through C‐type lectin Langerin, whereas subepithelial DCs express DC‐SIGN, which facilitates HIV‐1 transmission to T cells. As there is currently no HIV‐1 vaccine available, microbicides provide an alternative strategy to limit HIV‐1 spread. However, research into the function of LCs is hampered by the low availability and donor differences. Here, we set out to investigate whether LCs derived from the Mutz‐3 cell line (Mu‐LCs) provide a valuable tool to investigate the role of LCs in HIV‐1 transmission and identify suitable potential microbicides. We demonstrate that Mu‐LCs phenotypically resemble human primary LCs; Mu‐LCs do not transmit HIV‐1 efficiently, and inhibition of Langerin enhances HIV‐1 transmission to T cells. We show that carbohydrate structures blocking DC‐SIGN but not Langerin are potential microbicides, as they prevent HIV‐1 transmission by DCs but do not affect the antiviral function of LCs. Therefore, Mu‐LCs are a suitable model to investigate the role of LCs in HIV‐1 transmission and to screen potential microbicides.

Surgery-induced reactive oxygen species enhance colon carcinoma cell binding by disrupting the liver endothelial cell lining

Objective Resection of primary colorectal cancer is associated with enhanced risk of development of liver metastases. It was previously demonstrated that surgery initiated an early inflammatory response resulting in elevated tumour cell adhesion in the liver. Because reactive oxygen species (ROS) are shown to be produced and released during surgery, the effects of ROS on the liver vascular lining and tumour cell adhesion were investigated. Methods Human endothelial cell monolayers (human umbilical vein endothelial cells (HUVECs) and human microvascular endothelial cells of the lung (HMEC-1s)) were exposed to ROS production, after which electrical impedance, cellular integrity and tumour cell adhesion were investigated. Furthermore, surgery-induced tumour cell adhesion as well as the role of ROS and liver macrophages (Kupffer cells) in this process were studied in vivo. Results Production of ROS decreased cellular impedance of endothelial monolayers dramatically. Moreover, formation of intercellular gaps in endothelial monolayers was observed, exposing subendothelial extracellular matrix (ECM) on which colon carcinoma cells adhered via integrin molecules. Endothelial damage was, however, prevented in the presence of ROS-scavenging enzymes. Additionally, surgery induced downregulation of both rat and human liver tight junction molecules. Treatment of rats with the ROS scavenger edaravone prevented surgery-induced tumour cell adhesion and downregulation of tight junction proteins in the liver. Interestingly, depletion of Kupffer cells prior to surgery significantly reduced the numbers of adhered tumour cells and prevented disruption of expression of tight junction proteins. Conclusions In this study it is shown that surgery-induced ROS production by macrophages damages the vascular lining by downregulating tight junction proteins. This leads to exposure of ECM, to which circulating tumour cells bind. In light of this, perioperative therapeutic intervention, preventing surgery-induced inflammatory reactions, may reduce the risk of developing liver metastases, thereby improving the clinical outcome of patients with colorectal cancer.

Macrophages mediate colon carcinoma cell adhesion in the rat liver after exposure to lipopolysaccharide

The surgical resection of primary colorectal cancer is associated with an enhanced risk of liver metastases. Moreover, bacterial translocation or anastomic leakage during resection has been shown to correlate with a poor long-term surgical outcome, suggesting that bacterial products may contribute to the formation of metastases. Driven by these premises, we investigated the role of the bacterial product lipopolysaccharide (LPS) in the generation of liver metastases. Intraperitoneal injection of LPS led to enhanced tumor-cell adhesion to the rat liver as early as 1.5 h post-administration. Furthermore, a rapid loss of the expression of the tight junction protein zonula occludens-1 (ZO-1) was observed, suggesting that LPS disrupts the integrity of the microvasculature. LPS addition to endothelial-macrophage co-cultures damaged endothelial monolayers and caused the formation of intercellular gaps, which was accompanied by increased tumor-cell adhesion. These results suggest that macrophages are involved in the endothelial damage resulting from exposure to LPS. Interestingly, the expression levels of of ZO-1 were not affected by LPS treatment in rats in which liver macrophages had been depleted as well as in rats that had been treated with a reactive oxygen species (ROS) scavenger. In both settings, decreased tumor-cell adhesion was observed. Taken together, our findings indicate that LPS induces ROS release by macrophages, resulting in the damage of the vascular lining of the liver and hence allowing increased tumor-cell adherence. Thus, peri-operative treatments that prevent the activation of macrophages and—as a consequence—limit endothelial damage and tumor-cell adhesion may significantly improve the long-term outcome of cancer patients undergoing surgical tumor resection.