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The DC-HIL/syndecan-4 pathway inhibits human allogeneic T-cell responses
Abstract
T-cell activation is regulated by binding of ligands on APC to corresponding receptors on T cells. In mice, we discovered that binding of DC-HIL on APC to syndecan-4 (SD-4) on activated T cells potently inhibits T-cell activation. In humans, we now show that DC-HIL also binds to SD-4 on activated T cells through recognition of its heparinase-sensitive saccharide moiety. DC-HIL blocks anti-CD3-induced T-cell responses, reducing secretion of pro-inflammatory cytokines and blocking entry into the S phase of the cell cycle. Binding of DC-HIL phosphorylates SD-4's intracellular tyrosine and serine residues. Anti-SD-4 Ab mimics the ability of DC-HIL to attenuate anti-CD3 response more potently than Ab directed against other inhibitory receptors (CTLA-4 or programmed cell death-1). Among leukocytes, DC-HIL is expressed highest by CD14(+) monocytes and this expression can be upregulated markedly by TGF-beta. Among APC, DC-HIL is expressed highest by epidermal Langerhans cells, an immature type of dendritic cells. Finally, the level of DC-HIL expression on CD14(+) monocytes correlates inversely with allostimulatory capacity, such that treatment with TGF-beta reduced this capacity, whereas knocking down the DC-HIL gene augmented it. Our findings indicate that the DC-HIL/SD-4 pathway can be manipulated to treat T-cell-driven disorders in humans.
... Interestingly, most of the top enriched pathways in KEGG analysis, including lysosome [35][36][37] , proteoglycans in cancer 38 , focal adhesion 39 , ECM-receptor interaction 39 and PI3K-AKT signaling 40 had been reported for their correlation with GPNMB signaling. GPNMB is a transmembrane glycoprotein involved in various processes, including immune modulation 41,42 . However, the notion that GPNMB enhances the pathogenicity of obesity 41,43 or limits obesity-associated inflammation 42 is controversial. ...
... GPNMB is a transmembrane glycoprotein involved in various processes, including immune modulation 41,42 . However, the notion that GPNMB enhances the pathogenicity of obesity 41,43 or limits obesity-associated inflammation 42 is controversial. Therefore, we investigated the functional role of GPNMB in UHRF1 KO cells. ...
... Accumulating evidence suggests that GPNMB and TGF-β expression are interrelated 41,45 . To confirm this, we treated 3T3-L1 cells with recombinant GPNMB and analyzed the expression of TGF-β. ...
The E3 ubiquitin-ligase UHRF1 is an epigenetic regulator coordinating DNA methylation and histone modifications. However, little is known about how it regulates adipogenesis or metabolism. In this study, we discovered that UHRF1 is a key regulatory factor for adipogenesis, and we identified the altered molecular pathways that UHRF1 targets. Using CRISPR/Cas9-based knockout strategies, we discovered the whole transcriptomic changes upon UHRF1 deletion. Bioinformatics analyses revealed that key adipogenesis regulators such PPAR-γ and C/EBP-α were suppressed, whereas TGF-β signaling and fibrosis markers were upregulated in UHRF1-depleted differentiating adipocytes. Furthermore, UHRF1-depleted cells showed upregulated expression and secretion of TGF-β1, as well as the glycoprotein GPNMB. Treating differentiating preadipocytes with recombinant GPNMB led to an increase in TGF-β protein and secretion levels, which was accompanied by an increase in secretion of fibrosis markers such as MMP13 and a reduction in adipogenic conversion potential. Conversely, UHRF1 overexpression studies in human cells demonstrated downregulated levels of GPNMB and TGF-β, and enhanced adipogenic potential. In conclusion, our data show that UHRF1 positively regulates 3T3-L1 adipogenesis and limits fibrosis by suppressing GPNMB and TGF-β signaling cascade, highlighting the potential relevance of UHRF1 and its targets to the clinical management of obesity and linked metabolic disorders.
... GPNMB and adaptive immunity Impairment of T cell activation, proliferation and infiltration. Syndecan-4 (SD-4) is a transmembrane heparan-sulfate (HS) proteoglycan expressed on the surface of activated CD4 + and CD8 + T cells and has been identified as a ligand of GPNMB [42]. Activated T cells appear to express unique HS epitopes on SD-4 because GPNMB does not bind to B cells, despite their constitutive expression of SD-4 at high levels [43,44]. ...
... The GPNMB/SD-4 axis is emerging as a potent co-inhibitory pathway mediating T cell suppression. Binding of myeloid cell-derived GPNMB to the saccharide moiety of SD-4 on activated human or murine T cells strongly blocked anti-CD3-induced or APC-induced TCR-dependent primary and secondary CD4 + and CD8 + T cell responses, evidenced by decreased activation, proliferation and effector proinflammatory cytokine production [42]. Importantly, the inhibitory function of GPNMB on T cell activation is dependent on its PKD domain, which can fold into an immunoglobulin (Ig)-like tertiary structure required for binding to and inhibiting T cells [45]. ...
... Among leukocytes, GPNMB is constitutively and highly expressed by CD14 + monocytes, which possess APC capacity [42]. Transforming growth factor (TGF)-β stimulation further elevated GPNMB expression on CD14 + monocytes, which led to decreased IL-2 production by CD4 + T cells, an effect that was abolished by a knockdown of GPNMB in CD14 + monocytes [42]. ...
The immune system is comprised of both innate and adaptive immune cells, which, in the context of cancer, collectively function to eliminate tumor cells. However, tumors can actively sculpt the immune landscape to favor the establishment of an immunosuppressive microenvironment, which promotes tumor growth and progression to metastatic disease. Glycoprotein-NMB (GPNMB) is a transmembrane glycoprotein that is overexpressed in a variety of cancers. It can promote primary tumor growth and metastasis, and GPNMB expression correlates with poor prognosis and shorter recurrence-free survival in patients. There is growing evidence supporting an immunosuppressive role for GPNMB in the context of malignancy. This review provides a description of the emerging roles of GPNMB as an inducer of immunosuppression, with a particular focus on its role in mediating cancer progression by restraining pro-inflammatory innate and adaptive immune responses.
... Although previous work indicated that GPNMB is an anti-metastatic glycoprotein, recent studies question the function of GPNMB in cancer, because GPNMB appears to have a protective role in some types of cancers whereas it may promote metastasis in others (24). In the past years, GPNMB has been studied in breast cancer, whose role has been controversial for years, but most of the evidence suggests that GPNMB is associated with cancer aggressiveness, as it promotes cancer metastasis (42), angiogenesis (27) and immune suppression (40,44,45), enhancing tumor migration and invasion. An antibody anti-GPNMB (called CDX-011 or glembatumumab vedotin) has been evaluated in phase I/II trials as a treatment for advanced breast cancer and melanoma (46)(47)(48). ...
... Endogenous GPNMB prevents T cells from entering the cell cycle, resulting in the inhibition of its activation and proliferation, while GPNMB knockdown T cells display enhanced capacity for immunostimulation (30). Further studies showed that inhibition of T cell activation by GPNMB is dependent on the syndecan-4 pathway (78) and that GPNMB expression regulates lymphocyte allostimullatory capacity, which means the capability of producing antibodies (45). Several studies agree that GPNMB inhibits T cells, which indicates that GPNMB can weaken the immunological response, which is related to both onset and resolution of inflammation. ...
... Syndecan-4 is expressed in some activated T cells and presents specific heparan sulfates that bind to GPNMB. This interaction leads to a reduction of proinflammatory cytokine secretion and blocks T cells from entering the S phase of the cell cycle (45). ...
Inflammation is a response to a lesion in the tissue or infection. This process occurs in a specific manner in the central nervous system and is called neuroinflammation, which is involved in neurodegenerative diseases. GPNMB, an endogenous glycoprotein, has been recently related to inflammation and neuroinflammation. GPNMB is highly expressed in macrophages and microglia, which are cells involved with innate immune response in the periphery and the brain, respectively. Some studies have shown increased levels of GPNMB in pro-inflammatory conditions, such as LPS treatment, and in pathological conditions, such as neurodegenerative diseases and cancer. However, the role of GPNMB in inflammation is still not clear. Even though most studies suggest that GPNMB might have an anti-inflammatory role by promoting inflammation resolution, there is evidence that GPNMB could be pro-inflammatory. In this review, we gather and discuss the published evidence regarding this interaction.
... The length of human GPNMB is either 560 aa [72] or 572 aa [80]. Alternative splicing at the exon 6/7 results in a full length or a truncated version lacking 36 nucleotides/12 aa after aa 339, which is not reported for mouse or rat Gpnmb [89,90]. The splice site is located in the prolinerich region. ...
... The splice site is located in the prolinerich region. The shorter version can exhibit higher potency of inhibiting activated T cells [89]. Mouse and rat Gpnmb permanently contain the proline-serine rich insertion of 14 or 16 aa, respectively, which might account for improved melanosome handling [76]. ...
... Apart from macrophages, the second group of antigen-presenting cells are dendritic cells. Gpnmb is highly expressed in almost all subsets of dendritic cells of several species [74,85,89,132,134,145,146,149,150]. ...
Das globale Phänomen Übergewicht erhöht das Risiko für die Entwicklung von Diabetes, Atherosklerose und Herz-Kreislauf-Erkrankungen. Diese sind assoziiert mit der Expression des Transmembranproteins Glycoprotein nonmetastatic melanoma protein b (Gpnmb), das von Makrophagen und dendritischen Zellen exprimiert wird. Wir haben die Rolle von Gpnmb in genetisch- und diät-induzierter Atherosklerose sowie in diät-induzierter Adipositas in Gpnmb-Knockout- und Wildtyp-Mäusen untersucht. Körpergewicht und Blutfette wurden in beiden Erkrankungen nicht von Gpnmb beeinflusst. Gpnmb wurde in Makrophagen von atherosklerotischen Läsionen stark exprimiert, jedoch hatte das Fehlen von Gpnmb keinen Einfluss auf die Größe der Aortenläsion. In Übergewicht konnten wir dagegen einen größeren Effekt von Gpnmb detektieren. Gpnmb hatte einen positiven Einfluss auf den Insulin- und Glukoseplasmaspiegel sowie auf die Leberfibrose bei adipösen Mäusen. Gpnmb-Knockout-Tiere besaßen mehr Makrophagen im epididymalen Fettgewebe. Da Gpnmb in den entsprechenden Makrophagen von Wildtyp-Mäusen stark exprimiert wurde, könnte Gpnmb eine abschwächende Rolle auf Entzündungen des Fettgewebes haben. Dies wird durch in-vitro-Daten bestätigt, wo Gpnmb hauptsächlich in reparativen, TGFβ-stimulierten Makrophagen exprimiert wurde. Diese Expression führte jedoch nur zu einer leichten entzündungshemmenden Wirkung. Eine weitere Aufgabe von Makrophagen, die Autophagie, wurde durch Gpnmb nicht beeinflusst. Dies ist überraschend, da die Expression, die Freisetzung und der Abbau von Gpnmb durch Bafilomycin, einem Inhibitor des letzten Schritts der Autophagie, auf einzigartige Weise erhöht wurde. Zusammenfassend ist die Gpnmb-Expression in voll ausgereiften Makrophagen stark induziert und kann durch lysosomale Hemmung weiter gesteigert werden. Bei Adipositas verhindert Gpnmb die Entwicklung einer Insulinresistenz, möglicherweise durch Dämpfung der Entzündung des Fettgewebes.
... GPNMB can be stimulated by a variety of growth factors and cytokines, including IFNγ (in bone marrow-derived macrophages, myelogenous leukemia cell line k562, lung epithelial cell line CCL-64, and CD11b + Gr1 + cells), [46][47][48] IL1β (in CD11b + Gr1 + cells), 47 IL10 (in human monocyte-derived dendritic cells), 39 TGFβ (CD14 + monocytes), 49 and granulocyte-macrophage-colony stimulating factors (GM-CSF, in T47D breast cancer cells). 48 Other stimulants include LPS (microglia cells, RAW264.7 macrophages, and bone marrowderived macrophages) 46,50,51 and α-melanocyte-stimulating hormone (in melanoma cells). ...
... The extracellular domain of GPNMB on antigen-presenting cells suppresses T-cell activation and proliferation by binding to heparan sulfate-like structures on syndecan-4 on activated T cells. 49,[60][61][62][63][64][65][66] The critical role of GPNMB-syndecan-4 interaction between antigen-presenting cells and T cells has been suggested in different disease models, such as graft-versus-host disease, experimental autoimmune encephalomyelitis (EAE), and cancer. 47,[62][63][64]66 The antigen-presenting cells that express high levels of GPNMB include CD14 + monocytes and dendritic cells. ...
... 10,79 It is also expressed in other cell types of the skin, including keratinocytes and Langerhans cells. 13,49 Melanosomes are intracellular vesicles that produce, package, and store melanin in melanocytes. GPNMB is localized in early, stage III, and stage IV melanosomes, with relatively higher amounts detected in mature melanosomes. ...
The glycoprotein nonmetastatic melanoma protein B (GPNMB, also known as osteoactivin) is highly expressed in many cell types and regulates the homeostasis in various tissues. In different physiological contexts, it functions as a melanosome‐associated protein, membrane‐bound surface receptor, soluble ligand, or adhesion molecule. Therefore, GPNMB is involved in cell differentiation, migration, inflammation, metabolism, and neuroprotection. Because of its various involvement in different physiological conditions, GPNMB has been implicated in many diseases, including cancer, neurological disorders, and more recently immune‐mediated diseases. This review summarizes the regulation and function of GPNMB in normal physiology, and discusses the involvement of GPNMB in disease conditions with a particular focus on its potential role and therapeutic implications in autoimmunity.
... Blocking GPNMB by antibodies restores the integrity of T cells, attenuates tumor growth, and increases interferon-gamma levels in the tumor microenvironment 31 . The interaction between Syndecan-4 and GPNMB further leads to a reduction of proinflammatory cytokine secretion and hinders T cells from entering the S phase of the cell cycle 32 . Following these lines of evidence, a mouse MC38 colon adenocarcinoma cell line that was made resistant to anti-PD-1, transcriptomic profiles revealed that Gpnmb was significantly upregulated in anti-PD-1 therapy-resistant compared to -sensitive tumors. ...
... Furthermore, deletion of Gpnmb in resistant cells successfully restored sensitivity to anti-PD-1 treatment in vivo 33 . It has also been shown that higher circulating GPNMB blood levels of patients with advanced non-small cell lung cancer and malignant melanoma were associated with resistance to immune checkpoint blockade 32,34 . Taking together our results and the studies described here, GPNMB could be a marker of poor prognosis in PDS. ...
Pleomorphic dermal sarcomas are infrequent neoplastic skin tumors, manifesting in regions of the skin exposed to ultraviolet radiation. Diagnosing the entity can be challenging and therapeutic options are limited. We analyzed 20 samples of normal healthy skin tissue (SNT), 27 malignant melanomas (MM), 20 cutaneous squamous cell carcinomas (cSCC), and 24 pleomorphic dermal sarcomas (PDS) using mass spectrometry. We explored a potential cell of origin in PDS and validated our findings using publicly available single-cell sequencing data. By correlating tumor purity (TP), inferred by both RNA- and DNA-sequencing, to protein abundance, we found that fibroblasts shared most of the proteins correlating to TP. This observation could also be made using publicly available SNT single cell sequencing data. Moreover, we studied relevant pathways of receptor/ligand (R/L) interactions. Analysis of R/L interactions revealed distinct pathways in cSCC, MM and PDS, with a prominent role of PDGFRB-PDGFD R/L interactions and upregulation of PI3K/AKT signaling pathway. By studying differentially expressed proteins between cSCC and PDS, markers such as MAP1B could differentiate between these two entities. To this end, we studied proteins associated with immunosuppression in PDS, uncovering that immunologically cold PDS cases shared a “negative regulation of interferon-gamma signaling” according to overrepresentation analysis.
... Besides, GPNMB of in ltrating immune cells such as macrophages and dendritic cells, can inhibit T-cell activation or activate naive T-cells [16][17][18][19]. Also, GPNMB was detected highest in dendritic cells of antigen-presenting cells (APC), and it can bind to syndecan-4 of activated T cells to reduce T cells activation [20]. ...
... In melanoma, breast cancer, gliomas, prostate cancer, lung cancer, and bladder cancer, It was reported that high expression of GPNMB could promote cancer cell progression and invasion, and downregulate cancer cell apoptosis [24][25][38][39][40][41][42][43]. In colon cancer, tumor-in ltrating immune cells such as T cells could reduce tumor progression and the density, distribution of immune cells could haveadvantages in predicting survival status than TNM or Duke's classi cation [20]. The previous study reported thatTh2 cells, T helper cell in ltration could produce antitumor immunity and impacted the survival status of cancer patients [48]. ...
Background
Glycoprotein non-metastatic gene B (GPNMB) can regulate tumor progression by interacting with T cell function. However, the association between GPNMB and tumor-infiltrating immune cells and prognosis of various cancers is poorly understood.
Methods
We use the Oncomine and TIMER database to investigate GPNMB expression in multiple tumors. The PrognoScan database, Kaplan-Meier plotter are used to analyze tumor prognosis of GPNMB. R packages are used to performed multivariable cox regression analysis. We use TIMER and GEPIA database to explore the association between GPNMB expression and tumor immune infiltration levels, and immune cell markers. GPNMB related transcription factors and transcription-target networks are investigated via TTRUST database and GeneMANIA .
Results
A high level of GPNMB expression was significantly associated with poor prognosis in stomach adenocarcinoma (STAD). While, a high level of GPNMB expression was significantly associated with favorable prognosis in lung adenocarcinoma (LUAD). Besides, GPNMB expression levels can impact the prognosis in STAD and LUAD patients with lymph node metastasis. Moreover, GPNMB expression level has significant relationships with B cells, CD8 + T cells, CD4 + T cells, macrophages, neutrophils, and DCs infiltrating levels in STAD and LUAD. Besides, various immune gene markers of STAD and LUAD are significantly related to GPNMB expression. In addition, the GPNMB related transcription factors are MITF and TP53. The transcript-target networks are mainly responsible for signal transduction in response to DNA damage, DNA damage response, signal transduction by p53 class mediator, mitotic G1 DNA damage checkpoint, G1 DNA damage checkpoint.
Conclusions
These results indicate that GPNMB is significantly associated with prognosis and immune infiltrating levels in various cancers patients, especially in STAD, LUAD patients. Multiple immune gene markers of STAD and LUAD are significantly related to GPNMB expression, especially monocyte, macrophage polarization, and functional T cells gene markers. Our study signifies that GPNMB plays an essential role in prognosis prediction and immune infiltration of STAD and LUAD.
... Recently, the type I transmembrane receptor GPNMB (Glycoprotein NMB, DC-associated transmembrane protein (DC-HIL), osteoactivin), expressed on APC, was shown to strongly inhibit responses of CD4 + and CD8 + T cells by binding to its ligand syndecan-4 (SD-4) [14][15][16][17][18]. We previously demonstrated that primary human moDC moderately express GPNMB and dramatically upregulate its expression if generated in the presence of the cytokine IL-10, a main suppressor of cellular immunity, but notably also when exposed to imatinib, nilotinib or dasatinib [19,20]. ...
... (Figure 7 and Additional file 7: Figure S7). Chung et al. previously reported SD-4 to be the T cell ligand through which GPNMB mediates its negative coregulatory function [15][16][17][18]. Therefore, we assessed the specific role of GPNMB upregulation upon MK2206 treatment by addition of recombinant ligand SD-4. ...
Dendritic cells (DC) are the most potent antigen-presenting cells (APC) with the unique ability to activate naïve T cells and to initiate and maintain primary immune responses. Immunosuppressive and anti-inflammatory stimuli on DC such as the cytokine IL-10 suppress the activity of the transcription factor NF-κB what results in downregulation of costimulatory molecules, MHC and cytokine production. Glycoprotein NMB (GPNMB) is a transmembrane protein, which acts as a coinhibitory molecule strongly inhibiting T cell responses if present on APC. Interestingly, its expression on human monocyte-derived dendritic cells (moDC) is dramatically upregulated upon treatment with IL-10 but also by the BCR-ABL tyrosine kinase inhibitors (TKI) imatinib, nilotinib or dasatinib used for the treatment of chronic myeloid leukemia (CML). However, the molecular mechanisms responsible for GPNMB overexpression are yet unknown.
The immunosuppressive cytokine IL-10 and the BCR-ABL TKI imatinib or nilotinib, that were examined here, concordantly inhibit the PI3K/Akt signaling pathway, thereby activating the downstream serine/threonine protein kinase GSK3ß, and subsequently the microphthalmia-associated transcription factor (MITF) that is phosphorylated and translocated into the nucleus. Treatment of moDC with a small molecule inhibitor of MITF activity reduced the expression of GPNMB at the level of mRNA and protein, indicating that GPNMB expression is in fact facilitated by MITF activation. In line with these findings, PI3K/Akt inhibition was found to result in GPNMB overexpression accompanied by reduced stimulatory capacity of moDC in mixed lymphocyte reactions (MLR) with allogeneic T cells that could be restored by addition of the GPNMB T cell ligand syndecan-4 (SD-4).
In summary, imatinib, nilotinib or IL-10 congruently inhibit the PI3K/Akt signaling pathway thereby activating MITF in moDC, resulting in a tolerogenic phenotype. These findings extend current knowledge on the molecular mechanisms balancing activating and inhibitory signals in human DC and may facilitate the targeted manipulation of T cell responses in the context of DC-based immunotherapeutic interventions.
... We discovered new coinhibitors in DC-associated heparan sulfate proteoglycan-dependent integrin ligand (DC-HIL) on APCs and syndecan-4 (SD-4) on activated (but not resting) T cells (5,6). DC-HIL belongs to the Ig receptor superfamily (95-120 kDa) expressed constitutively by epidermal Langerhans cells, DCs, macrophages, and other monocytes (7). Binding of DC-HIL to SD-4 + T cells strongly inhibits T cell activation triggered via the TCR (5,7). ...
... DC-HIL belongs to the Ig receptor superfamily (95-120 kDa) expressed constitutively by epidermal Langerhans cells, DCs, macrophages, and other monocytes (7). Binding of DC-HIL to SD-4 + T cells strongly inhibits T cell activation triggered via the TCR (5,7). Blocking such binding through soluble DC-HIL receptor or anti-SD-4 Ab augments delayed-type hypersensitivity responses (6,8), and infusion of SD-4 2/2 T cells into sublethally gamma-irradiated allogeneic mice worsened acute graft-versus-host disease (9). ...
Having discovered that the dendritic cell (DC)-associated heparan sulfate proteoglycan-dependent integrin ligand (DC-HIL) receptor on APCs inhibits T cell activation by binding to syndecan-4 (SD-4) on T cells, we hypothesized that the DC-HIL/SD-4 pathway may regulate autoimmune responses. Using experimental autoimmune encephalomyelitis (EAE) as a disease model, we noted an increase in SD-4(+) T cells in lymphoid organs of wild-type (WT) mice immunized for EAE. The autoimmune disease was also more severely induced (clinically, histologically, and immunophenotypically) in mice knocked out for SD-4 compared with WT cohorts. Moreover, infusion of SD-4(-/-) naive T cells during EAE induction into Rag2(-/-) mice also led to increased severity of EAE in these animals. Similar to SD-4 on T cells, DC-HIL expression was upregulated on myeloid cells during EAE induction, with CD11b(+)Gr-1(+) myeloid-derived suppressor cells (MDSCs) as the most expanded population and most potent T cell suppressor among the myeloid cells examined. The critical role of DC-HIL was supported by DC-HIL gene deletion or anti-DC-HIL treatment, which abrogated T cell suppressor activity of MDSCs, and also by DC-HIL activation inducing MDSC expression of IFN-γ, NO, and reactive oxygen species. Akin to SD-4(-/-) mice, DC-HIL(-/-) mice manifested exacerbated EAE. Adoptive transfer of MDSCs from EAE-affected WT mice into DC-HIL(-/-) mice reduced EAE severity to the level of EAE-immunized WT mice, an outcome that was precluded by depleting DC-HIL(+) cells from the infused MDSC preparation. Our findings indicate that the DC-HIL/SD-4 pathway regulates autoimmune responses by mediating the T cell suppressor function of MDSCs.
... Our results emphasize the importance of the glioma environment for the tumor promoting effect of GPNMB. Indeed, GPNMB has been reported to impair T cell proliferation, activation and extravasation by binding Syndecan-4, a mechanism relevant in immunity [25][26][27][28][42][43][44]. This activity of GPNMB could explain our in vivo observation, namely the reduced levels of Foxp3 + T regs and PD-1 + T cells in the glioma tissue combined with increased levels of cytotoxic cells, CD8 + cells and proliferating T cells. ...
Tumor-associated microglia and blood-derived macrophages (TAMs) play a central role in modulating the immune suppressive microenvironment in glioma. Here, we show that GPNMB is predominantly expressed by TAMs in human glioblastoma multiforme and the murine RCAS-PDGFb high grade glioma model. Loss of GPNMB in the in vivo tumor microenvironment results in significantly smaller tumor volumes and generates a pro-inflammatory innate and adaptive immune cell microenvironment. The impact of host-derived GPNMB on tumor growth was confirmed in two distinct murine glioma cell lines in organotypic brain slices from GPNMB-KO and control mice. Using published data bases of human glioma, the elevated levels in TAMs could be confirmed and the GPNMB expression correlated with a poorer survival.
Supplementary Information
The online version contains supplementary material available at 10.1186/s40478-024-01754-7.
... Even though developing a vaccine against GPNMB positive cells seems to be beneficial, the challenge remains to only deplete cells with the highest expression of the protein but not the cells expressing it at a physiological level 63 . Moreover, GPNMB has protective roles in the body by contributing to the resolution of inflammation and by inhibiting T cells activation 64,65 . Therefore, the development of this vaccine in clinical trials could be more complicated than in in vivo experiment, given the diverse physiological functions of GPNMB in normal cells. ...
The involvement of cellular senescence in the initiation and propagation of diseases is clearly characterized, making the elimination of senescent cells essential to treat age-related diseases. The development of senolytic drugs demonstrated that targeting these cells limits the deterioration of patients’ condition, by inducing apoptosis. Nevertheless, the first generations of senolytics which has been developed displayed their activities through specific mechanisms and demonstrated several limitations during clinical development. However, the rational to eliminate senescent cells remains evident, with the necessity to develop specific therapies in a context of diseases and tissues. The evolutions in the field of drug discovery open the way to a new generation of senolytic therapies, such as immunological approaches (CAR-T cells, Antibody-Drug Conjugated or vaccines), which require preliminary steps of research to identify markers specifically expressed on senescent cells, demonstrating promising specific effects. Currently, the preclinical development of these strategies appears more challenging to avoid strong side effects, but the expected results are commensurate with patients’ hopes for treatments. In this review, we highlight the fact that the classical senolytic approach based on drug repurposing display limited efficacy and probably reached its limits in term of clinical development. The recent development of more complex therapies and the extension of interest in the domain of senescence in different fields of research allow to extend the possibility to discover powerful therapies. The future of age-related diseases treatment is linked to the development of new approaches based on cell therapy or immunotherapy to offer the best treatment for patients.
... Meanwhile, it participates the differentiation of immune cells into macrophages, lymphocytes, DCs and associated with osteoblasts and osteoclasts in bone and can also functionally impair T cell activation [14,15]. ...
Small cell lung cancer (SCLC) is notorious for its early and frequent metastases, which contribute to it as a recalcitrant malignancy. Previously, we found 4 significantly top up-regulated genes in lymph node (LN) metastases tissue when compared with primary tumor by HTG Edge-seq. Among them, Glycoprotein non-metastatic melanoma protein B (GPNMB) was highly expressed in the metastatic lymph node in SCLC. The aim of this study was to investigate the prognostic value and cellular functions of GPNMB in SCLC. Plasma concentration of GPNMB was performed by enzyme-linked immunosorbent assay (ELISA). Clinical characteristic and Overall survival (OS) based on GPNMB concentration was evaluated. Invasion, migration, proliferation and apoptosis ability of SCLC cells were evaluated using migration, matrigel invasion assays, CCK8 assay and flow cytometry respectively. We found that GPNMB was significantly up-regulated in LN than primary SCLC lesions by HTG Edge-seq. 88 patients with SCLC were included in this study. Plasma GPNMB concentration was significantly higher in patients with extensive disease than local disease (P = 0.043). Baseline plasma GPNMB expression was higher in liver metastasis when compared with non-liver metastasis (P = 0.0148), bone metastasis (P = 0.031), brain metastasis (P = 0.035) and non-organ metastasis (P = 0.0045). Patients with high baseline plasma GPNMB had shorter overall survival (OS) (10.32 vs. 16.10 months, P = 0.0299). Importantly, both univariate and multivariate analyses indicated GPNMB higher expression was an independent biomarker for OS (P = 0.040, HR = 0.444). We also found that inhibit GPNMB expression by siRNA significantly suppressed SCLC metastatic and proliferation ability whereas increased apoptosis ability and caused a significant downregulation of vimentin and upregulation of E-cadherin. Our results suggest that expression of GPNMB is associated with metastasis and poor prognosis, and might be served as a novel potential therapeutic target in SCLC.
... The next highest correlation was observed for TCL1A (R = 0.82; FDR = .04) ( Figure 4B), which is a critical player in several lymphoid malignances, and has been demonstrated to act as a coactivator to augment the activity of AKT kinases (32), Figure S5), also had well-described roles in regulating immunity (34)(35)(36)(37). We also noted that SHC phosphorylation level on Tyr317 (SHC-pY317) was negatively correlated with ROR (R = -0.75; ...
Despite impressive antitumor efficacy of programmed cell death 1 (PD-1) inhibitors, this inhibition can induce mild to severe autoimmune toxicities, termed immune-related adverse events (irAEs). Yet, predictive pretreatment biomarkers for irAEs development across cancer types remain elusive. We first assessed cellular and molecular factors. To determine factors predicting the risk of irAEs for anti–PD-1 immunotherapy across multiple cancer types, an integrative analysis of cellular and molecular factors from 9104 patients across 21 cancer types and 4865522 postmarketing adverse event reports retrieved from adverse event reporting system was then performed. Accuracy of predictions was quantified as Pearson correlation coefficient determined using leave-one-out cross-validation. Independent validation sets included small cell lung cancer and melanoma cohorts. Out of 4865522 eligible adverse events reports, 10412 cases received anti–PD-1 monotherapy, of which, 2997 (28.78%) exhibited at least one irAE. Among established immunogenomic factors, dendritic cells (DC) abundance showed the strongest correlation with irAEs risk, followed by tumor mutational burden (TMB). Further predictive accuracy was achieved by DC and TMB in combination with CD4⁺ naive T-cells abundance, and then validated in the small cell lung cancer cohort. Additionally, global screening of multiomics data identified 11 novel predictors of irAEs. Of these, IRF4 showed the highest correlation. Best predictive performance was observed in the IRF4 – TCL1A – SHC-pY317 trivariate model. Associations of IRF4 and TCL1A expression with irAEs development were verified in the melanoma cohort receiving immune checkpoint inhibitors. Collectively, pretreatment cellular and molecular irAEs-associated features as well as their combinations are identified regardless of cancer types. These findings may deepen our knowledge of irAEs pathogenesis and, ultimately, aid in early detection of high-risk patients and management of irAEs.
... Therefore, high GPNMB secretion in GAMs was capable of inducing protumorigenic phenotypes of GAMs [109]. Studies have demonstrated that GPNMB was capable of blocking T cell stimulation via direct cell-cell intercommunication of antigen-presenting cells (APCs) and T cells resulting in immunosuppressive milieu in gliomas ( Figure 2) [110,111]. SPP1 was also recognized as a ligand for CD44, and SPP1-CD44 communication augmented the stemness of CD44 secretory glioma-stimulatory cells ( Figure 2) [112]. Furthermore, it was established that GAMs and not any other cells in the tumor microenvironment were the principal source of SPP1 secretion in glioma [112]. ...
Gliomas are mixed solid tumors composed of both neoplastic and nonneoplastic cells. In glioma microenvironment, the most common nonneoplastic and infiltrating cells are macrophages and microglia. Microglia are the exact phagocytes of the central nervous system, whereas macrophages are myeloid immune cells that are depicted with ardent phagocytosis. Microglia are heterogeneously located in almost all nonoverlapping sections of the brain as well as the spinal cord, while macrophages are derived from circulating monocytes. Microglia and macrophages utilize a variety of receptors for the detection of molecules, particles, and cells that they engulf. Both microglia and peripheral macrophages interact directly with vessels both in the periphery of and within the tumor. In glioma milieu, normal human astrocytes, glioma cells, and microglia all exhibited the ability of phagocytosing glioma cells and precisely apoptotic tumor cells. Also, microglia and macrophages are robustly triggered by the glioma via the expression of chemoattractants such as monocyte chemoattractant protein, stromal-derived factor-1, and macrophage-colony stimulating factor. Glioma-associated microglia and/or macrophages positively correlated with glioma invasiveness, immunosuppression, and patients' poor outcome, making these cells a suitable target for immunotherapeutic schemes.
... DC-HIL inhibits proliferation of T cells dependent on T cell receptors and blocks reactivation of T cells. DC-HIL could be upregulated by TGF-b (74,75). Microglia gain APC-like properties by the expression of CD11c, CD86 and intracellular adherin molecule 1, stimulated by neuroinflammation. ...
Amyotrophic lateral sclerosis (ALS), an incurable, devastating condition of the central nervous system, is characterized by selective destruction of motoneurons with an important contribution of innate and adaptative immunity. Microglia and immune cells are key players in neuroinflammation and active participants in ALS pathogenesis. Recent experiments in animal models have shown that microglia display both neuroprotective and neurotoxic properties, depending on the stage of disease progression and cytokine secretion. A better knowledge of the interactions between T cells and microglia in the immunopathogenesis of ALS is desirable for the development of ALS therapeutic strategies.
... 13,14 Glycoprotein NMB interacts with syndecan-4, which is expressed on the surface of T cells, through the PKD, and this interaction suppresses T-cell activation and proliferation. [15][16][17][18][19] So far, little is known about the function of the KLD in GPNMB. Therefore, in the present study, we clarified the contribution of the KLD to the tumorigenic function of GPNMB. ...
Glycoprotein NMB (GPNMB) is highly expressed in many types of malignant tumors and thought to be a poor prognostic factor in those cancers, including breast cancer. GPNMB is a type IA transmembrane protein that has a long extracellular domain (ECD) and a short intracellular domain (ICD). In general, the ECD of a protein is involved in protein‐protein or protein‐carbohydrate interactions, while the ICD is important for intracellular signaling. We previously reported that GPNMB contributes to the initiation and malignant progression of breast cancer through the hemi‐immunoreceptor tyrosine‐based activation motif (hemITAM) in its ICD. Furthermore, we demonstrated that the tyrosine residue in hemITAM is involved in induction of the stem‐like properties of breast cancer cells. However, the contribution of the ECD to its tumorigenic function has yet to be fully elucidated. In this study, we focused on the region, the so‐called kringle‐like domain (KLD), that is conserved among species, and made a deletion mutant, GPNMB(ΔKLD). Enhanced expression of wildtype (WT) GPNMB induced sphere and tumor formation in breast epithelial cells; in contrast, GPNMB(ΔKLD) lacked these activities without affecting its molecular properties such as subcellular localization, Src‐induced tyrosine phosphorylation at least in overexpression experiments, and homo‐oligomerization. GPNMB(ΔKLD) also lost its cell‐migration promoting activity, even though it reduced E‐cadherin expression. Although the interaction partner binding to KLD has not yet been identified, we found that the KLD of GPNMB has an important role for its tumorigenic potential.
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... DC-expressed, membrane bound GPNMB is found to bind to T-cells, thereby inhibiting the proliferation of CD4 + and CD8 + T-cells and the secretion of IL-2 [142]. Syndecan-4, an heparan sulfate proteoglycan (HSPG) containing membrane protein on activated T-cells, has been identified as primary ligand for GPNMB [143][144][145]. Binding of GPNMB to syndecan-4 is thought to take place in two steps: initial binding via the extracellular arginylglycylaspartic acid (RGD-) domain facilitates PKD-dependent binding [109]. ...
Several diseases are caused by inherited defects in lysosomes, the so-called lysosomal storage disorders (LSDs). In some of these LSDs, tissue macrophages transform into prominent storage cells, as is the case in Gaucher disease. Here, macrophages become the characteristic Gaucher cells filled with lysosomes laden with glucosylceramide, because of their impaired enzymatic degradation. Biomarkers of Gaucher cells were actively searched, particularly after the development of costly therapies based on enzyme supplementation and substrate reduction. Proteins selectively expressed by storage macrophages and secreted into the circulation were identified, among which glycoprotein non-metastatic protein B (GPNMB). This review focusses on the emerging potential of GPNMB as a biomarker of stressed macrophages in LSDs as well as in acquired pathologies accompanied by an excessive lysosomal substrate load in macrophages.
... Patients with other autoimmune conditions, including SLE, were excluded. Peripheral blood mononuclear cells were incubated with 10 mg/ml mouse anti-human DC-HIL monoclonal antibody (3D5) (Chung et al., 2009) or isotype-control mouse IgG and 1 mg/ml phycoerythrin-antimouse IgG. Cells were further stained with 2.5 mg/ml BD Horizon V500-conjugated antieHLA-DR (BD Biosciences, San Jose, CA) and phycoerythrin-Cy7-anti-CD14 antibody (BioLegend, San Diego, CA), and analyzed by flow cytometry. ...
... For example, DC-HIL inhibits T cell receptor (TCR)-dependent proliferation of T cells and blocks reactivation of T cells previously activated by APCs (66). Among APCs, DC-HIL expression is the highest on epidermal Langerhans cells (LCs) and can be upregulated by TGFβ (67). Furthermore, it has been shown that mutations in DC-HIL result in compromised ocular immunity and aberrant adaptive immune responses to self-antigen (68). ...
In recent years, many paradigms concerning central nervous system (CNS) immunology have been challenged and shifted, including the discovery of CNS-draining lymphatic vessels, the origin and functional diversity of microglia, the impact of T cells on CNS immunological homeostasis and the role of neuroinflammation in neurodegenerative diseases. In parallel, antigen presentation outside the CNS has revealed the vital role of antigen-presenting cells in maintaining tolerance toward self-proteins, thwarting auto-immunity. Here, we review recent findings that unite these shifted paradigms of microglial functioning, antigen presentation, and CNS-directed T cell activation, focusing on common neurodegenerative diseases. It provides an important update on CNS adaptive immunity, novel targets, and a concept of the microglia T-cell equilibrium.
... When this interaction is perturbed, either by targeting DC-HIL on myeloid cells or Sdc4 on T cells, there is an increase in T cell activation and EAE is exacerbated [128]. Furthermore, the suppressive effect of Sdc4 on T cell activation was demonstrated by direct ligation [129]. Given the opposing effects of MK and DC-HIL/Sdc4 perturbation in that model, it raises the question of the functional consequences of MK binding to Sdc4 on T cells. ...
Significance:
The extracellular matrix (ECM) is the non-cellular component secreted by cells and present within all tissues and organs. The ECM provides the structural support required for tissue integrity and also contributes to diseases, including cancer. Many diseases rich in ECM are characterized by changes in reactive oxygen species (ROS) levels that have been shown to have important context-dependent functions. Recent Advances: Many studies have found that the ECM affects ROS production through integrins. The activation of integrins by ECM ligands results in stimulation of multiple pathways that can generate ROS. Further, control of ECM-integrin interaction by matricellular proteins is an underappreciated pathway that functions as an ROS rheostat in remodeling tissues.
Critical issues:
A better understanding of how the ECM affects the generation of intracellular ROS is required for advances in the development of therapeutic strategies that affect or exploit oxidative stress.
Future directions:
Targeting ROS generation can be therapeutic or promote disease progression in a context-dependent manner. Many ECM proteins can impact ROS generation. However, given the breadth of different proteins that constitute the ECM and the cell surface receptors that interact with ECM proteins, there are likely many tissue and microenvironmental-specific ROS-generating pathways that have yet to be investigated in depth. Identifying canonical pathways of ECM-induced ROS generation should be a priority for the field.
... When this interaction is perturbed, either by targeting DC-HIL on myeloid cells or Sdc4 on T cells, there is an increase in T cell activation and EAE is exacerbated [128]. Furthermore, the suppressive effect of Sdc4 on T cell activation was demonstrated by direct ligation [129]. Given the opposing effects of MK and DC-HIL/Sdc4 perturbation in that model, it raises the question of the functional consequences of MK binding to Sdc4 on T cells. ...
Cytokines are pivotal in the generation and resolution of the inflammatory response. The midkine/pleiotrophin (MK/PTN) family of cytokines, composed of just two members, was discovered as heparin-binding neurite outgrowth-promoting factors. Since their discovery, expression of this cytokine family has been reported in a wide array of inflammatory diseases and cancer. In this minireview, we will discuss the emerging appreciation of the functions of the MK/PTN family in the immune system, which include promoting lymphocyte survival, sculpting myeloid cell phenotype, driving immune cell chemotaxis, and maintaining hematopoiesis.
... Curiously, the same group showed after that DC-HIL could also recognize dermatophytes, as T. rubrum and M. audouinii, but not non-dermatophytes as C. albicans, playing a positive role in cytokine production and DC activation [63]. The authors postulated that since DC-HIL is highly expressed by epidermal Langerhans cells [64], it would be a key receptor in the immune response to dermatophytes. ...
Innate immunity is the host first line of defense against pathogens. However, only in recent years, we are beginning to better understand the ways it operates. A key player is this branch of the immune response that are the phagocytes, as macrophages, dendritic cells and neutrophils. These cells act as sentinels, employing specialized receptors in the sensing of invaders and host injury, and readily responding to them by production of inflammatory mediators. They afford protection not only by ingesting and destroying pathogens, but also by providing a suitable biochemical environment that shapes the adaptive response. In this review, we aim to present a broad perspective about the role of phagocytes in dermatophytosis, focusing on the mechanisms possibly involved in protective and non-protective responses. A full understanding of how phagocytes fit in the pathogenesis of these infections may open the venue for the development of new and more effective therapeutic approaches.
... PBMCs (5 x 10 5 cells) isolated from the blood samples were treated with human IgG (1 µg/ml for blocking) and incubated with 10 µg/ml 3D5 mouse anti-human DC-HIL mAb (Chung et al., 2009) ...
Psoriasis vulgaris is an inflammatory skin disease caused by hyper-activated T-cells regulated by positive and negative mechanisms; while the former has been much studied, but the latter has not. We studied the regulatory mechanism mediated by myeloid-derived suppressor cells (MDSCs), especially having shown that MDSCs expanded in melanoma patients express DC-HIL, a critical mediator of T-cell suppressor function. We examined expansion of DC-HIL+ MDSCs in psoriasis and characterized their functional properties. Frequency of DC-HIL+ monocytic MDSCs (CD14+HLA-DRno/low) in blood and skin was markedly increased in psoriatic patients vs. healthy controls, but there was no statistically significant relationship with disease severity (PASI score). Blood DC-HIL+ MDSC levels in the untreated patients were significantly higher than the treated patients. Compared to melanoma-derived MDSCs, psoriatic MDSCs exhibited significantly reduced suppressor function, and were less dependent on DC-HIL, but capable of inhibiting proliferation and IFN-γ and IL-17 responses of autologous T-cells. Psoriatic MDSCs were functionally diverse among patients in their ability to suppress allogeneic T-cells and use of either IL-17/arginase I or IFN-γ/iNOS axis as suppressor mechanisms. Thus DC-HIL+ MDSCs are expanded in psoriasis patients, and their mechanistic heterogeneity and relative functional deficiency may contribute to the development of psoriasis.
... Gpnmb is induced in macrophages and RAW264.7 cells by interferon-γ (IFN-γ ) and lipopolysaccharide, and negatively regulates the production of cytokines IL-6 and IL-12 26 . The binding between antigen presenting dendritic cells and T cells negatively regulated the T cell activation 23,27 and this process is mediated by the interaction between Gpnmb on DCs and syndecan-4 on T cells in allogenic 28,29 and autoimmune 30 responses. ...
Nonalcoholic fatty liver disease (NAFLD) is the most common cause of chronic liver disease worldwide. Gpnmb is classified as a type 1 membrane protein and its soluble form is secreted by ADAM10-mediated cleavage. Gpnmb mRNA was found in the Kupffer cells and white adipose tissues (WATs) and its upregulation in obesity was recently found. Here, we generated aP2 promoter-driven Gpnmb transgenic (Tg) mice and the overexpression of Gpnmb ameliorated the fat accumulation and fibrosis of the liver in diet-induced obesity model. Soluble form of Gpnmb in sera was elevated in Gpnmb Tg mice and Gpnmb concentrated in hepatic macrophages and stellate cells interacted with calnexin, which resulted in the reduction of oxidative stress. In the patients with non-alcoholic steatohepatitis, serum soluble GPNMB concentrations were higher compared with the patients with simple steatosis. The GPNMB is a promising biomarker and therapeutic target for the development and progression of NAFLD in obesity.
... Other mechanisms have become increasingly studied, such as the recent finding of the prominent role of the DC-associated heparan sulfate proteoglycandependent integrin ligand (DC-HIL)/Syndecan-4 pathway in the suppression of T cells [82]. DC-HIL belongs to the Ig receptor superfamily and is expressed on Langerhans cells, DCs, macrophages [83] and CD11b + Gr-1 + MDSCs [82]. Binding of DC-HIL to syndecan 4 on the activated T cell surface strongly inhibits T cell activation and proliferation. ...
The knowledge of the immune system elements and their relationship with other tissues, organs and systems are key approximations for the resolution of many immune-related disorders. The control of the immune response and/or its modulation from the pro-inflammatory to the anti-inflammatory response is being deeply studied in the field. In the last years, the study of myeloid-derived suppressor cells (MDSCs), a group of immature myeloid cells with a high suppressive activity on T cells has been extensively addressed in cancer. In contrast, their role in neuroimmune diseases is far from being totally understood. In this review, we will summarize data about MDSCs coming from the study of neuroinflammatory diseases in general and their potential role in multiple sclerosis, in order to introduce the putative use of this extraordinary promising cell type for future cell-based therapies. This article is part of a Special Issue entitled: Neuro inflammation: A common denominator for stroke, multiple sclerosis and Alzheimer's disease, guest edited by Helga de Vries and Markus Swaninger.
... These results indicate that GPNMB may directly prevent muscular pathology in SOD1 G93A mice and should be considered a therapeutic target for ALS. required for the differentiation and function of osteoblasts (Selim et al., 2003;Abdelmagid et al., 2008) and osteoclasts (Ripoll et al., 2008;Sheng et al., 2008), inhibition of T-cell activation (Chung et al., 2007(Chung et al., , 2009, and invasion and metastasis by several cancers (Onaga et al., 2003;Rich et al., 2003;Kuan et al., 2006;Tse et al., 2006;Rose et al., 2007;Williams et al., 2010). GPNMB expression significantly increases with disease progression in the spinal cords of patients with ALS and in transgenic mice that serve as a model of ALS (Tanaka et al., 2012). ...
Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by progressive loss of motor neurons and subsequent muscular atrophy. The quality of life of patients with ALS is significantly improved by ameliorating muscular symptoms. We previously reported that glycoprotein nonmetastatic melanoma protein B (GPNMB; osteoactivin) might serve as a target for ALS therapy. In the present study, superoxide dismutase 1/glycine residue 93 changed to alanine (SOD1(G93A) ) transgenic mice were used as a model of ALS. Expression of the C-terminal fragment of GPNMB was increased in the skeletal muscles of SOD1(G93A) mice and patients with sporadic ALS. SOD1(G93A) /GPNMB transgenic mice were generated to determine whether GPNMB expression ameliorates muscular symptoms. The weight and cross-sectional area of the gastrocnemius muscle, number and cross-sectional area of myofibers, and denervation of neuromuscular junctions were ameliorated in SOD1(G93A) /GPNMB vs. SOD1(G93A) mice. Furthermore, direct injection of a GPNMB expression plasmid into the gastrocnemius muscle of SOD1(G93A) mice increased the numbers of myofibers and prevented myofiber atrophy. These findings suggest that GPNMB directly affects skeletal muscle and prevents muscular pathology in SOD1(G93A) mice and may therefore serve as a target for therapy of ALS. © 2015 Wiley Periodicals, Inc.
© 2015 Wiley Periodicals, Inc.
... These include histones [21], cyclophilin B [22,23] and the chemokine RANTES [24]. Binding of a dendritic cell ligand (dendritic cell-associated heparan sulfate proteoglycan-dependent integrin ligand) to syndecan-4 on T cells reduces T cell proliferation and IL-2 production [25][26][27][28][29]. Binding of thrombospondin-1 to CD47 on Jurkat cells also has inhibitory effects [30]. ...
Proteoglycans are components of the extracellular matrix and have roles in brain development and responses to injury. Connective tissue components are known to be major inhibitors of remyelination in mouse models of demyelination and are found at the border of active demyelinating lesions in Multiple Sclerosis. Surfen (bis 2-methyl, 4- amino, 6-quinolyl amide) is a small molecule antagonist previously shown to bind preferentially to heparan sulfate and related proteoglycans.
We have previously reported that surfen reduces T cell proliferation
in vivo
and
in vitro
. Here we report the effects of surfen on an
in vivo
model of demyelination and its effects on macrophage function
in vitro
. Demyelination was induced by injecting the detergent lysolecithin into the spinal cord dorsal columns of adult C57Bl/6 mice. Relative to vehicle treated mice, co-injection of surfen (100 μM) with lysolecithin reduced total lesion area seven days post-injection. Because macrophages dominate these lesions and influence remyelination, murine bone marrow derived macrophages were assessed using assays of chemotaxis and phagocytosis. Macrophages chemotaxis was increased in response to surfen (10 μM) relative to vehicle by approximately 15% (p<0.05). Phagocytosis of E. coli was not affected by surfen.
These effects of surfen on experimental demyelination and macrophage function suggest that proteoglycan binding may promote aspects of myelin repair relevant to Multiple Sclerosis.
... Thus, high GPNMB expression in GAMs could participate in the modulation of the pro-tumorigenic phenotype of GAMs. Furthermore, GPNMB has been shown to inhibit T cell activation via direct cell-cell interaction of antigen-presenting cells and T cells, and could thus contribute to the immunosuppressive milieu in gliomas [50,51,52]. Finally, anti-Gpnmb antibodies conjugated with a cytotoxic agent are under investigation for the treatment of malignant glioma, breast cancer, and cutaneous melanoma [53,54,55]. ...
Malignant glioma belong to the most aggressive neoplasms in humans with no successful treatment available. Patients suffering from glioblastoma multiforme (GBM), the highest-grade glioma, have an average survival time of only around one year after diagnosis. Both microglia and peripheral macrophages/monocytes accumulate within and around glioma, but fail to exert effective anti-tumor activity and even support tumor growth. Here we use microarray analysis to compare the expression profiles of glioma-associated microglia/macrophages and naive control cells. Samples were generated from CD11b<sup>+</sup> MACS-isolated cells from naïve and GL261-implanted C57BL/6 mouse brains. Around 1000 genes were more than 2-fold up- or downregulated in glioma-associated microglia/macrophages when compared to control cells. A comparison with published data sets of M1, M2a,b,c-polarized macrophages revealed a gene expression pattern that has only partial overlap with any of the M1 or M2 gene expression patterns. Sampl
... Cross-linking of SDC-4 by antibodies also attenuated anti-CD3-induced T-cell responses in vitro. In contrast, ablation of SDC-4 by specific antibodies, knockdown of SDC-4, or treatment with soluble SDC-4 led to an enhanced T-cell response to antigen, in a DC-HILdependent manner, and in both human and mouse systems (Chung et al., 2007a;2009). Importantly, by knocking down DC-HIL in a B16-F10 melanoma cell line, Tomihari et al. (2010) showed that melanoma-associated DC-HIL could inhibit melanoma-reactive T cells and promote tumor growth in vivo, and in an SDC-4-dependent manner. ...
An immune-suppressive role of myeloid-derived suppressor cells (MDSCs) in melanoma has long been speculated, whereas molecular mechanisms underlying this role are not well understood. Here, Chung and colleagues show that dendritic cell-associated, heparan sulfate proteoglycans-dependent integrin ligand (DC-HIL), a cell surface immune-modulatory molecule, is highly expressed on tumor-associated MDSCs. Genetic ablation or antibody blockade of DC-HIL delays the growth of transplantable B16 melanoma in syngeneic mice, which is accompanied by enhanced antitumor T-cell activities. These findings support a role for DC-HIL in immune evasion within the melanoma microenvironment.
Reducing the activity of cytokines and leukocyte extravasation is an emerging therapeutic strategy to limit tissue-damaging inflammatory responses and restore immune homeostasis in inflammatory diseases. Proteoglycans embedded in the vascular endothelial glycocalyx, which regulate the activity of cytokines to restrict the inflammatory response in physiological conditions, are proteolytically cleaved in inflammatory diseases. Here we critically review the potential of proteolytically shed, soluble vascular endothelial glycocalyx proteoglycans to modulate pathological inflammatory responses. Soluble forms of the proteoglycans syndecan-1, syndecan-3 and biglycan exert beneficial anti-inflammatory effects by the removal of chemokines, suppression of proinflammatory cytokine expression and leukocyte migration, and induction of autophagy of proinflammatory M1 macrophages. By contrast, soluble versikine and decorin enhance proinflammatory responses by increasing inflammatory cytokine synthesis and leukocyte migration. Endogenous syndecan-2 and mimecan exert proinflammatory effects, syndecan-4 and perlecan mediate beneficial anti-inflammatory effects and glypican regulates Hh and Wnt signaling pathways involved in systemic inflammatory responses. Taken together, targeting the vascular endothelial glycocalyx-derived, soluble syndecan-1, syndecan-2, syndecan-3, syndecan-4, biglycan, versikine, mimecan, perlecan, glypican and decorin might be a potential therapeutic strategy to suppress overstimulated cytokine and leukocyte responses in inflammatory diseases.
Aortic aneurysms, aortic dissections and acute aortic syndromes are disastrous conditions, putting the affected patient in immediate fatal risk. To facilitate further understanding of these conditions and to support early diagnosis and prognostic stratification are ground laying goals of angiology and cardiovascular surgery. This study focusses on “lingering” Stanford-type-B-dissections in stable condition allowing for a conservative approach. The aim was to clarify whether within the examined series of patients an indication of a possible proteomic signature for the course of aortic events can be found. A total of 50 aortic patient serum samples and 20 controls were examined. All serum samples were processed, labelled in a TMT-16plex, and measured in a shot-gun explorative, bottomup LC-MS/MS approach for proteomic analysis. The generated data was statistically evaluated to identify differentially abundant proteins between the conditional groups. To depicture our postulated gain of severity within the course of aortic diseases, an alleged time series is modulated. Throughout the groups it was searched for a protein intensity trend with either upward or downward tendency, mimicking the changes in stability of the aortic vessel. In this manner two supervised clusters were identified to match the expected trends that held interesting biological motives within the context of aortic disease and instability in the Top GO enrichment analysis. Further investigation of the subgroup of “lingering” type-B-dissections, including the clinical data, uncovered four significant proteins trending along increasing instability of the aorta within this group using a limma analysis. Three of these proteins that were found to be increased with increasing severity, were also found significant when comparing aneurysm, acute aortic syndromes, and surgically stabilised dissections. The discovered proteins Transaldolase and Histone H4 could be identified as signs of a cellular destruction imprint in term of aortic weakening. The third protein Vinculin, with its function in cell stability and cell contact is especially interesting in context of aortic dissections, since following an intimal tear, blood is flowing within the media of the vessel in-between cells that would normally be connected via various cell-cell connection entities that vinculin is naturally a part of. With this first statement of aortic stability signature proteins, we may in the long term be able to influence the daily clinical routine, because these identified signature proteins bear potential to help diagnose aortic diseases as well as assess patients’ necessity for urgent treatment comparable to an early warning system. This might eventually contribute to clearer risk stratification for patients, because the decision for treatment could be pillowed and opted for as long as the patient is still in better clinical constitution.
Cancer immunoediting progresses through elimination, equilibrium and escape. Each of these phases is characterized by breaching, remodeling and rebuilding tissue planes and structural barriers that engage extracellular matrix (ECM) components, in particular matrix proteoglycans. Some of the signals emanating from matrix proteoglycan remodeling are readily co-opted by the growing tumor to sustain an environment of tumor-promoting and immune-suppressive inflammation. Yet other matrix-derived cues can be viewed as part of a homeostatic response by the host, aiming to eliminate the tumor and restore tissue integrity. These latter signals may be harnessed for therapeutic purposes to tip the polarity of the tumor immune milieu towards anti-cancer immunity. In this review, we attempt to showcase the importance and complexity of matrix proteoglycan signaling in both cancer-restraining and cancer-promoting inflammation. We propose that the era of matrix diagnostics and therapeutics for cancer is fast approaching the clinic.
Die Einführung der Tyrosinkinaseinhibitoren (TKIs) stellt einen Meilenstein der Hämatoonkologie im 21. Jahrhundert dar. Im Kontext der chronisch myeloischen Leukämie (CML) konnte das BCR-Abl-Fusionstranskript als pathognomonisches Korrelat identifiziert werden. Dessen pharmakologische Hemmung über bestimmte TKIs korrelierte nicht nur mit hervorragenden Remissionsraten, sondern auch mit einer deutlich besseren Verträglichkeit. Dasatinib verfügt als Zweitgenerations-TKI neben einer hohen Bindungsaffinität zum BCR-Abl-Fusionstranskript als Multi-TKI auch über weitere Zielstrukturen, zu welchen u.a. die Kinasen der Src-Familie (SFKs) gehören. Diese übernehmen bei verschiedenen Immunzellen wichtige regulatorische Funktionen. Bei einem Teil der CML-Patienten kann die TKI-Therapie unterbrochen werden und es findet sich eine anhaltende Remission. Hierfür wurden immunmodulatorische Effekte der TKIs angenommen. Für Dasatinib konnten in vitro und in vivo immunmodulatorische Effekte, u.a. auf T- und NK-Zellen nachgewiesen werden. Im Rahmen dieser Arbeit sollten mögliche immunmodulatorische Effekte auf dendritische Zellen monozytärer Abstammung (moDZ) in vitro untersucht werden. Die Generierung von moDZs erfolgte mittels Zugabe von IL-4 und GM-CSF aus Monozyten gesunder Blutspender. Dasatinib wurde in klinisch relevanten Konzentrationen (10-50 nM) ab Beginn der DZ-Generierung zugegeben, für ausgewählte Experimente auch kurz vor Reifung mit LPS. Dabei wurde der Einfluss von Dasatinib auf die Generierung von moDZs sowie wichtige Funktionen unreifer (Endozytose) und reifer DZs (Expression von zellulären und humoralen Effektorfunktionen) analysiert. Dasatinib bewirkte in einer Konzentration von 50 nM eine verminderte Generierung von moDZs aus Monozyten. Phänotypisch zeigte sich dabei eine verminderte Expression des DZ-typischen Markers CD1a bei einer persistierenden Expression des monozytären Markers CD14. Parallel fand sich auch eine Population CD1a/CD14-koexprimierender Zellen. Daneben konnte auch eine Apoptosesteigerung unter Dasatinib nachgewiesen werden. In Bezug auf die Makropinozytose FITC-konjugierter Dextranpartikel wurde keine relevante Modulation beschrieben. Nach Reifung mit LPS bewirkte Dasatinib 50 nM eine verminderte Expression der costimulatorischen Oberflächenmarker CD80 und CD86, sowie eine verminderte Sekretion von IL-12. Zusammenfassend zeigen unter Dasatinib generierte moDZs Eigenschaften, welche auch bei regulatorischen DZs beschrieben wurden. Eine Behandlung mit Dasatinib könnte folglich Immunantworten negativ modulieren.
The development of senomorphic drugs to attenuate the senescent phenotype and senolytics to clear pro-inflammatory senescent cells to treat aging-associated disorders is being hotly pursued. The effort is complicated by the fact that senescent cells play a constructive role in some cellular processes such as tissue repair and wound healing. However, concerns about efficacy, which senescent cells to target, and unwanted side effects have created potential roadblocks. Chimeric Antigen Receptor (CAR) T cells directed against urokinase-type plasminogen activator receptor (uPAR), which is expressed on at least a subset of senescent cells (SC) in atherosclerotic plaques and fibrotic livers, removed SC and improved glucose metabolism. A conventional vaccine targeting CD153-expressing senescent T-cells, also improved glucose metabolism in obese mice. Recent work to selectively target senescent cells associated with several pathologies has resulted in the creation of a peptide vaccine that primarily targets endothelial cells expressing high levels of GPNMB, recently identified as a biomarker of senescence. The vaccine reduces atherosclerotic plaque burden and metabolic dysfunction such as glucose intolerance in mouse models of obesity and atherosclerosis. For translation to humans the activity of the vaccine will need to be tightly controlled, as the target, GPNMB has multiple roles in normal physiology including acting to inhibit and possibly resolve inflammation. A promising alternative approach would be to use passive immunization with a monoclonal antibody directed against GPNMB.
Acquired resistance to the antitumor activity of antibodies targeting the programmed death 1 (PD-1): programmed death ligand 1 (PD-L1) immune checkpoint in various types of cancers has increasingly been observed during treatment. To gain insight into the molecular mechanism underlying anti-PD-1 therapy resistance, we developed a mouse MC38 colon adenocarcinoma cell line that was made resistant to anti-PD-1 treatment through repeated in vivo selection. We compared the transcriptomic profiles of anti-PD-1 therapy-resistant and -sensitive tumors using RNA sequencing analysis. The immunosuppressive molecule transmembrane glycoprotein NMB (GPNMB) was significantly upregulated in resistant tumor cells, as determined using quantitative real-time polymerase chain reaction and immunofluorescence analyses. Furthermore, deletion of GPNMB in resistant cells successfully restored sensitivity to anti-PD-1 treatment in vivo. Collectively, our results indicate that tumors may develop resistance to anti-PD-1 therapy by upregulating their expression of the immunosuppressive molecule GPNMB. Furthermore, GPNMB is a potential, targetable biomarker for monitoring adaptive resistance to therapeutic PD-1 blockade, and identification of this immunosuppressive molecule may be a breakthrough for new therapies.
Obesity can cause a chronic, low-grade inflammation, which is a critical step in the development of type II diabetes and cardiovascular diseases. Inflammation is associated with the expression of glycoprotein nonmetastatic melanoma protein b (Gpnmb), which is mainly expressed by macrophages and dendritic cells. We generated a Gpnmb-knockout mouse line using Crispr-Cas9 to assess the role of Gpnmb in a diet-induced obesity. The absence of Gpnmb did not affect body weight gain and blood lipid parameters. While wildtype animals became obese but remained otherwise metabolically healthy, Gpnmb-knockout animals developed, in addition to obesity, symptoms of metabolic syndrome such as adipose tissue inflammation, insulin resistance and liver fibrosis. We observed a strong Gpnmb expression in adipose tissue macrophages in wildtype animals and a decreased expression of most macrophage-related genes independent of their inflammatory function. This was corroborated by in vitro data showing that Gpnmb was mostly expressed by reparative macrophages while only pro-inflammatory stimuli induced shedding of Gpnmb. The data suggest that Gpnmb is ameliorating adipose tissue inflammation independent of the polarization of macrophages. Taken together, the data suggest an immune-balancing function of Gpnmb that could delay the metabolic damage caused by the induction of obesity.
The gene GPNMB is known to play roles in phagocytosis and tissue repair, and is upregulated in microglia in many mouse models of neurodegenerative disease as well as in human patients. Nearby genomic variants are associated with both elevated Parkinson's disease (PD) risk and higher expression of this gene, suggesting that inhibiting GPNMB activity might be protective in Parkinson's disease. We tested this hypothesis in three different mouse models of neurological diseases: a remyelination model and two models of alpha-synuclein pathology. We found that Gpnmb deletion had no effect on histological, cellular, behavioral, neurochemical or gene expression phenotypes in any of these models. These data suggest that Gpnmb does not play a major role in the development of pathology or functional defects in these models and that further work is necessary to study its role in the development or progression of Parkinson's disease.
Purpose:
Immune checkpoint inhibitors (ICI) benefit only a minority of treated cancer patients. Identification of biomarkers distinguishing responders and non-responders will improve management of cancer patients. Since the DC-HIL checkpoint differs from the PD1 pathway in expression and inhibitory mechanisms, we examined whether DC-HIL expression regulates ICI responsiveness.
Experimental design:
Plasma samples were collected from advanced non-small cell lung carcinoma (NSCLC) patients (n=76) at baseline and/or follow-up after ICI monotherapy. Blood soluble DC-HIL (sDC-HIL) was determined and analyzed for correlation with the early tumor response. To study the mechanisms, we measured effect of anti-DC-HIL vs. anti-PDL1 mAb on growth of mouse tumor cells in experimentally metastatic lung. Influence of DC-HIL to anti-PDL1 treatment was assessed by changes in tumor response after deletion of host-DC-HIL gene, injection of DC-HIL-expressing myeloid-derived suppressor cells (MDSC) or induction of sDC-HIL expression.
Results:
Non-responders expressed significantly higher levels of baseline sDC-HIL levels compared to responders. Among patients (n=28) for fluctuation with time, non-responders (14/15 cases) showed increasing or persistently elevated levels. Responders (12/13) had decreasing or persistently low levels. Among various tumors, B16 melanoma exhibited resistance to anti-PDL1, but responded to anti-DC-HIL mAb. Using B16 melanoma and LL2 lung cancer, we showed deletion of host-derived DC-HIL expression converted the resistant tumor to one responsive to anti-PDL1 mAb. The responsive state was revered by infusion of DC-HIL+MDSC or induction of sDC-HIL expression.
Conclusions:
sDC-HIL in the blood and probably DC-HIL receptor expressed by MDSC plays an important role in regulating response to ICI in advanced NSCLC.
Prolactin family 7, subfamily d, member 1 (Prl7d1), a member of the expanding prolactin family, is mainly expressed in the placental junctional zone (including trophoblast giant cells and spongiotrophoblast cells) with peak expression observed at 12 days postcoitum (dpc) in mice. Previous studies have shown that PRL7D1 is a key mediator of angiogenesis in vitro; however, its physiological roles in placental development in vivo have not been characterized. To address this issue, we deleted Prl7d1 in mice and demonstrated that its absence results in reduced litter size and fertility. Histologically, Prl7d1 mutants exhibited striking placental abnormalities at 12.5 dpc, including a reduction in the proportion of labyrinth layers and a significant increase in decidual natural killer cells, glycogen trophoblasts, and trophoblast giant cells in the junctional zone. Moreover, placentas from Prl7d1‐null mice displayed a thickened decidual spiral artery. Notably, these negative effects were more pronounced in male fetuses. Further RNA‐sequencing analysis showed that Prl7d1 deletion results in significant differences in the placental transcriptome profile between the two sexes of fetuses. Together, this study demonstrates that Prl7d1 possesses antiangiogenic properties in deciduas and inhibits the development of junctional zone, which potentially alters the functional capacity of the placenta to support optimal fetal growth. Moreover, of note, the role of Prl7d1 in the placenta is regulated in a fetal sex‐specific manner. Prolactin family 7, subfamily d, member 1 (Prl7d1) deficiency leads to disorders in multiple signaling pathways, and in turn, might result in placental structural defects. Moreover, significant differences in placental phenotypes and gene expression profiles, depending on the sex of the fetus, were observed.
Purpose:
Blocking the function of myeloid-derived suppressor cells (MDSCs) is an attractive approach for cancer immunotherapy. Having shown DC-HIL/GPNMB to be the T cell-inhibitory receptor mediating the suppressor function of MDSCs, we evaluated the potential of anti-DC-HIL monoclonal antibody (mAb) as a MDSC-targeting cancer treatment.
Experimental design:
Metastatic cancer patients (n=198) were analyzed by flow-cytometry for DC-HIL or PDL1 expression on blood CD14+HLA-DRno/lo MDSCs. Their suppressor function was assessed by in vitro co-culture with autologous T-cells, and the ability of anti-DC-HIL or anti-PDL1 mAb to reverse such function was determined. Tumor expression of these receptors was examined histologically, and the anti-tumor activity of the mAb was evaluated by attenuated growth of colon cancers in mice.
Results:
Metastatic cancer patients had high blood levels of DC-HIL+ MDSCs compared to healthy controls. Anti-DC-HIL mAb reversed the in vitro function in ~80% of cancer patients tested, particularly for colon cancer. Despite very low expression on blood MDSCs, anti-PDL1 mAb was as effective as anti-DC-HIL mAb in reversing MDSC function, a paradoxical phenomenon we found to be due to upregulated expression of PDL1 by T cell-derived IFN-Υ in co-cultures. DC-HIL is not expressed by colorectal cancer cells, but by CD14+ cells infiltrating the tumor. Finally, anti-DC-HIL mAb attenuated growth of pre-established colon tumors by reducing MDSCs and increasing IFN-Υ-secreting T-cells in the tumor microenvironment, with similar outcomes to anti-PDL1 mAb.
Conclusions:
Blocking DC-HIL function is a potentially useful treatment for at least colorectal cancer with high blood levels of DC-HIL+ MDSCs.
Autologous dendritic cells (DCs) loaded with cancer cell-derived lysates have become a promising tool in cancer immunotherapy. During the last decade, we demonstrated that vaccination of advanced melanoma patients with autologous tumor antigen presenting cells (TAPCells) loaded with an allogeneic heat shock-(HS-) conditioned melanoma cell-derived lysate (called TRIMEL) is able to induce an antitumor immune response associated with a prolonged patient survival. TRIMEL provides not only a broad spectrum of potential melanoma-associated antigens but also danger signals that are crucial in the induction of a committed mature DC phenotype. However, potential changes induced by heat conditioning on the proteome of TRIMEL are still unknown. The identification of newly or differentially expressed proteins under defined stress conditions is relevant for understanding the lysate immunogenicity. Here, we characterized the proteomic profile of TRIMEL in response to HS treatment. A quantitative label-free proteome analysis of over 2800 proteins was performed, with 91 proteins that were found to be regulated by HS treatment: 18 proteins were overexpressed and 73 underexpressed. Additionally, 32 proteins were only identified in the HS-treated TRIMEL and 26 in non HS-conditioned samples. One protein from the overexpressed group and two proteins from the HS-exclusive group were previously described as potential damage-associated molecular patterns (DAMPs). Some of the HS-induced proteins, such as haptoglobin, could be also considered as DAMPs and candidates for further immunological analysis in the establishment of new putative danger signals with immunostimulatory functions.
Objective:
Glycoprotein non-metastatic protein B (GPNMB) is a transmembrane glycoprotein that is expressed at higher levels in several malignant human tissues than those in matched normal tissues. Thus, GPNMB may serve as an attractive therapeutic target of cancer treatment. In this study, the prognostic value of GPNMB expression was examined in tumors derived from a cohort of patients with epithelial ovarian cancer (EOC).
Methods:
GPNMB expression in matched formalin-fixed and paraffin-embedded tissue samples was evaluated by immunohistochemistry (IHC), whereas GPNMB mRNA expression in fresh-frozen biopsy tissues was detected using real-time quantitative PCR (qPCR). Meanwhile, the correlations of GPNMB expression with the clinical characteristics of EOC were assessed. Besides, survival data were analysed using Kaplan-Meier and Cox regression analyses, respectively.
Results:
GPNMB expression was remarkably upregulated in EOC tissues compared with that in normal ovarian controls at both mRNA and protein levels. In addition, abundant GPNMB expression in EOC was correlated with the International Federation of Gynecology and Obstetrics (FIGO) stage (P < 0.001), residual tumor (P = 0.036), and lymph node metastasis (P = 0.004). Furthermore, results of univariate and multivariate analyses indicated that GPNMB expression level was an independent prognostic factor of the progression-free survival (PFS) and overall survival (OS) (P < 0.001 and P < 0.001, respectively) for EOC patients.
Conclusion:
Upregulated GPNMB levels in EOC patients are associated with dismal prognosis. Moreover, findings in the current study indicate that GPNMB is a potentially useful prognostic predictor of the therapeutic approaches for EOC.
Glycoprotein nonmetastatic melanoma protein B (GPNMB) has a neuroprotective effect against neuronal cell death caused by the accumulation of abnormal mutated proteins. It is known that the accumulation of pathological proteins induces endoplasmic-reticulum (ER) stress leading to cell damage. The aim of this study was to determine the role of GPNMB in the ER stress response. GPNMB was greatly up-regulated by thapsigargin-induced ER stress. Under the ER stress conditions, GPNMB relocated to the nucleus and specifically up-regulated expression of BiP at the mRNA level by promoting the BiP pre-mRNA splicing, not through the pathways initiated by the three major transducers of the unfolded protein response: IRE1, PERK, and ATF6. Furthermore, we found that the protein level of BiP and the infarction were increased and attenuated, respectively, in Gpnmb-transgenic mice after occlusion of the middle cerebral artery, in comparison with wild-type mice. Thus, our findings indicate that GPNMB enhances the BiP expression by promoting the splicing (thereby preventing cell death caused by ER stress) and could be a therapeutic target in ER stress-related disorders.
GPNMB has emerged as an immunomodulator and an important positive mediator of tumor progression and metastasis in numerous solid cancers. Tumor intrinsic GPNMB-mediated effects on cellular signaling, coupled with the ability of GPNMB to influence the primary tumor and metastatic microenvironments in a non-cell autonomous fashion, combine to augment malignant cancer phenotypes. In addition, GPNMB is often overexpressed in a variety of cancers, making it an attractive therapeutic target. In this regard, glembatumumab vedotin, an antibody-drug conjugate (ADC) that targets GPNMB, is currently in clinical trials as a single agent in multiple cancers.
In this review, we will describe the physiological functions of GPNMB in normal tissues and summarize the processes through which GPNMB augments tumor growth and metastasis. We will review the pre-clinical and clinical development of glembatumumab vedotin, evaluate on-going clinical trials, explore emerging opportunities for this agent in new disease indications and discuss exciting possibilities for this ADC in the context of combination therapies.
Introduction:
Osteoactivin (OA) was first discovered in an osteopetrotic rat model using mRNA differential display a decade ago and has been studied recently. OA in bone tissue can directly or indirectly regulate the differentiation of osteoblasts by influencing cell behaviours, such as proliferation and adhesion, as well as inducing serial signal cascades, which would be of great importance in the field of tissue engineering. The results of recent studies have further demonstrated that OA plays a critical role in the differentiation and function of cells, especially in bone formation and fracture healing.
Areas covered:
The discovery, structure, and function of OA as well as its therapeutic potential in tissue regeneration of bone defects, kidney injury, liver damage, and muscle atrophy.
Expert opinion:
OA has great potential in promoting the regeneration of damaged tissues, particularly bone tissue, which is supported by a large body of data. Future studies should focus on exploring the underlying mechanism of OA as well as pursuing the ideal form of OA-related regenerative medicine.
The development of monoclonal antibodies as cancer therapeutic agents has improved the outlook for many patients as evidenced by the number of such drugs that have been approved for oncologic indications [1]. Monoclonal antibodies allow selective killing of malignant cells (targeted therapy) with relative sparing of the normal tissues resulting in higher therapeutic efficacy and lower toxicity. The advances in the monoclonal antibody field have been expedited by the discovery of targets or pathways that are uniquely present or upregulated in cancer cells along with the advances in the technology used for the production of human or “humanized” reagents [2]. An additional strategy to enhance monoclonal antibody therapy is to utilize these reagents to selectively deliver radioisotopes (radioimmunotherapy) or cytotoxic agents (antibody–drug conjugates) to tumor cells [3, 4]. CDX-011 is a recently described antibody–drug conjugate in early clinical trials.
Objective: To investigate the differences of glycoprotein non-metastatic melanoma b (Gpnmb) expression between M1 and M2 bone marrow-derived macrophages (BMMψs) in mouse. Methods: Primary BMMψs were cultured and then identified by immunofluorescence staining for F4/80 and flow cytometry testing of CD11b. Interferon-γ and lipopolysaccharide were used to induce differentiation of BMMψs towards M1 macrophages and interleukin-4 was adopted to induce differentiation of M2 macrophages. Realtime PCR was performed to analyze mRNA expressions of tumor necrosis factor (TNF-α), inducible NO synthase (iNOS), macrophage mannose receptor (MMR), arginase-1 (Arg-1) and Gpnmb. Proteins of Gpnmb and MMR were detected by double immunofluorescence staining, Western blot and flow cytometry. Results: (1) Immunofluorescence staining showed high expression of F4/80 in BMMψs and flow cytometry results showed that CD11b was expressed in 92.7%±6.1% of BMMψs, suggesting that primary BMMψs were successfully cultured. (2) Compared with M0 BMMψs, mRNAs of TNF-α and iNOS were highly up-regulated in M1 BMMψs (both P < 0.01), and mRNAs of MMR and Arg-1 were highly up-regulated in M2 BMMψs (both P < 0.01), indicating that differentiation of BMMψs towards M1 and M2 BMMψs were successfully induced. (3) Expressions of Gpnmb mRNA and Gpnmb protein were predominantly up-regulated in M2 BMMψs in comparison with those in M0 and M1 BMMψs (both P < 0.01). Gpnmb and MMR were co-expressed in M2 BMMψs and 83.2%±9.7% of MMR positive BMMψs expressed Gpnmb. Conclusion: Gpnmb expression is significantly increased in M2 macrophages than that in M1 macrophages in vitro, indicating that Gpnmb which takes part in the differentiation of macrophages might be used as a marker for identification of M1 and M2 macrophages.
Although MSCs have been widely recognized to have therapeutic potential in the repair of injured or diseased tissues, it remains unclear how functional activities of mesenchymal stem cells (MSCs) are influenced by the surrounding inflammatory milieu at the site of tissue injury. Macrophages constitute an essential component of innate immunity and have been shown to exhibit a phenotypic plasticity in response to various stimuli, which play a central role in both acute inflammation and wound repair. Osteoactivin (OA)/Glycoprotein non-metastatic melanoma protein B (GPNMB), a transmembrane glycoprotein that plays a role in cell differentiation, survival and angiogenesis. The objective of this study was to investigate the potential role of OA/GPNMB in macrophage-induced MSC function. We found that reparative M2 macrophages express significantly greater levels of OA/GPNMB than pro-inflammatory M1 macrophages. Furthermore, using loss of function and rescue studies, we demonstrate that M2 macrophages-secreted OA/GPNMB positively regulates the viability, proliferation, and migration of MSCs. More importantly, we demonstrated that OA/GPNMB acts through ERK and AKT signaling pathways in MSCs via CD44, to induce these effects. Taken together, our results provide pivotal insight into the mechanism by which OA/GPNMB contributes to the tissue reparative phenotype of M2 macrophages and positively regulates functional activities of MSCs. This article is protected by copyright. All rights reserved.
Glycoprotein nonmelanoma protein B ( GPNMB , alias osteoactivin), a type I transmembrane glycoprotein, is cleaved by extracellular proteases, resulting in release of an extracellular fragment ( ECF ). GPNMB is widely expressed by neurons within the CNS , including the hippocampus; however, its function in the brain remains unknown. Here, we investigated the role of GPNMB in memory and learning by using transgenic (Tg) mice over‐expressing GPNMB (Tg mice on a BDF ‐1 background) and ECF ‐treated mice. In the hippocampus of both wild‐type and Tg mice, GPNMB was highly expressed in neurons and astrocytes. Tg mice exhibited memory improvements in two types of learning tasks but were impaired in a passive‐avoidance test. In Tg mice, the hippocampus displayed increased levels of the α‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionate receptor subunit GluA1. Intracerebroventricular administration of ECF (50 ng) to Institute of Cancer Research ( ICR ) mice also improved memory in a passive‐avoidance test and increased hippocampal GluA1 levels 24 h after treatment. In Tg mice and ECF (0.25 μg/mL)‐treated hippocampal slices, long‐term potentiation was promoted. These findings suggest that GPNMB may be a novel target for research on higher order brain functions. image
Glycoprotein nonmelanoma protein B (GPNMB) is widely expressed in neurons. We investigated the role of GPNMB on memory by using transgenic mice over‐expressing GPNMB (Tg) and GPNMB extracellular fragment (ECF)‐treated mice. Both mice exhibited memory improvement. In Tg mice, protein levels of phosphorylated α‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionate (AMPA) receptor subunit GluA1, CaMK2 (Ca ²⁺ /calmodulin‐dependent protein kinase 2), and GSK3β (glycogen synthase kinase 3β) were increased. Tg mice and ECF‐treated hippocampus promoted LTP. These findings suggest that GPNMB might become a novel target for research on higher order brain functions.
Skin contains receptors for various environmental factors. In this issue of the Journal, Busse et al. cloned a new olfactory receptor, OR2AT4, in keratinocytes. They show that the activation of OR2AT4 induces phosphorylation of extracellular signal-regulated kinases and p38 mitogen-activated protein kinases, and that it accelerates wound healing. OR2AT4 may be a promising candidate as a target in clinical drug development.
CD160, a glycosylphosphatidylinositol-anchored member of the immunoglobulin superfamily, is expressed on both cytolytic lymphocytes and some unstimulated CD4(+) T cells. Here we show that CD160 expression was increased after activation of human CD4(+) T cells and that crosslinking CD160 with monoclonal antibody strongly inhibited CD3- and CD28-mediated activation. We found that herpesvirus entry mediator (HVEM) was a ligand of CD160 that acted as a 'bidirectional switch' for T cell activation, producing a positive or negative outcome depending on the engagement of HVEM by CD160 and known HVEM ligands such as B and T lymphocyte attenuator (BTLA) and the T lymphocyte receptor LIGHT. Inhibition of CD4(+) T cell activation by HVEM-transfected cells was dependent on CD160 and BTLA; when the cysteine-rich domain 1 of HVEM was deleted, this inhibition was lost, resulting in strong T cell activation. CD160 thus serves as a negative regulator of CD4(+) T cell activation through its interaction with HVEM.
Langerhans cells (LCs) are dendritic cells (DCs) that are present in the epidermis, bronchi, and mucosae. Although LCs originate in bone marrow, little is known about their lineage of origin. In this study, we demonstrate that in vitro LCs may originate from monocytes. Adult peripheral blood CD14+ monocytes differentiate into LCs (CD1a+, E-cadherin+, cutaneous lymphocyte-associated antigen+, Birbeck granules+, Lag+) in the presence of granulocyte/macrophage colony-stimulating factor, interleukin 4, and transforming growth factor beta1 (TGF-beta1). This process occurs with virtually no cell proliferation and is not impaired by 30 Gy irradiation. Selection of monocyte subpopulations is ruled out since monocyte-derived DCs can further differentiate into LCs. Our data suggest that in vivo LC differentiation may be induced peripherally, from a nonproliferating myeloid precursor, i.e., the monocyte, in response to a TGF-beta1-rich microenvironment, as found in the skin and epithelia. Therefore, the monocyte may represent a circulating precursor critical to the immune response in vivo.
Syndecans are transmembrane proteoglycans capable of carrying both heparan and chondroitin sulfate chains. The cytoplasmic
tail of syndecan-4 was recently reported to undergoin vivo phosphorylation on Ser183 in the membrane-proximal part of the tail (Horowitz, A., and Simons, M. (1998)J. Biol. Chem. 273, 10914–10918). However, the functional consequences of this event remain unknown. The cytoplasmic tail of syndecan-4 is
known to undergo multimerization and to activate protein kinase Cα (PKCα), with both events depending on the presence of the
commonly occurring phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2). In the present investigation we found that phosphorylation of Ser183 produced a 10-fold reduction in the ability of syndecan-4 to activate PKCα, without affecting its ability to bind the PKC.
Because Ser183 is adjacent to positively charged lysine groups that resemble PIP2-binding regions in several other proteins, phosphorylation of this serine may affect the binding affinity of the syndecan-4
cytoplasmic tail to PIP2. We found that the Ser183-phosphorylated cytoplasmic tail of syndecan-4 has indeed a significantly lower affinity to PIP2 compared with the nonphosphorylated tail. Furthermore, Ser183phosphorylation abolished PIP2-dependent oligomerization of syndecan-4 cytoplasmic tails. We conclude that Ser183 phosphorylation regulates syndecan-4-dependent activation of PKCα by reducing the affinity to PIP2 and inhibiting the oligomerization of syndecan-4 cytoplasmic tails. These results further support the role of syndecan-4
in signal transduction in endothelial cells.
Following ligation of the TCR and costimulatory molecules such as CD28, T cells proliferate and secrete cytokines. Several other cell surface molecules have been identified that are capable of augmenting activation mediated via the TCR. These include CD2, CD27, CD40 ligand, and signaling lymphocytic activation molecule. Here, we have characterized the expression and function of CD148, a recently identified receptor-type protein tyrosine phosphatase. CD148 is expressed at low levels on resting T cells, but is up-regulated following in vitro activation. Cross-linking CD148 with immobilized anti-CD148 mAb induced vigorous proliferation of anti-CD3 mAb-activated, highly purified peripheral blood T cells in an IL-2-dependent, cyclosporin A-sensitive manner. This effect was greatest after 8 days of in vitro culture, suggesting that this molecule is involved in the latter stages of a T cell response. CD148-induced proliferation was significantly greater for CD8+ T cells than for CD4+ T cells. Thus, CD148 is a receptor-type protein tyrosine phosphatase involved in the activation of T lymphocytes.
Most cases of autosomal dominant polycystic kidney disease (ADPKD) are the result of mutations in the PKD1 gene. The PKD1 gene codes for a large cell-surface glycoprotein, polycystin-1, of unknown function, which, based on its predicted domain structure, may be involved in protein-protein and protein-carbohydrate interactions. Approximately 30% of polycystin-1 consists of 16 copies of a novel protein module called the PKD domain. Here we show that this domain has a beta-sandwich fold. Although this fold is common to a number of cell-surface modules, the PKD domain represents a distinct protein family. The tenth PKD domain of human and Fugu polycystin-1 show extensive conservation of surface residues suggesting that this region could be a ligand-binding site. This structure will allow the likely effects of missense mutations in a large part of the PKD1 gene to be determined.
We isolated a novel molecule (DC-HIL) expressed abundantly by the XS52 dendritic cell (DC) line and epidermal Langerhans cells,
but minimally by other cell lines. DC-HIL is a type I transmembrane protein that contains a heparin-binding motif and an integrin-recognition
motif, RGD, in its extracellular domain (ECD). A soluble fusion protein (DC-HIL-Fc) of the ECD and an immunoglobulin Fc bound
to the surface of an endothelial cell line (SVEC). This binding induced adhesion of SVEC to its immobilized form. Sulfated
polysaccharides (e.g. heparin and fucoidan) inhibited binding of soluble DC-HIL-Fc and adhesion of SVEC. By contrast, an integrin inhibitor (RGDS
tetramer) had no effect on binding to SVEC, but prevented adhesion of SVEC. This differential RGD requirement was confirmed
by the finding that DC-HIL-Fc mutant lacking the RGD motif can bind to SVEC but is unable to induce adhesion of SVEC. Furthermore,
DC-HIL appears to recognize directly these sulfated polysaccharides. These results suggest that DC-HIL binds to SVEC by recognizing
heparan sulfate proteoglycans on endothelial cells, thereby inducing adhesion of SVEC in an RGD-dependent manner. We propose
that DC-HIL serves as a DC-associated, heparan sulfate proteoglycan-dependent integrin ligand, which may be involved in transendothelial
migration of DC.
Programmed death I (PD-I)-deficient mice develop a variety of autoimmune-like diseases, which suggests that this immunoinhibitory receptor plays an important role in tolerance. We identify here PD-1 ligand 2 (PD-L2) as a second ligand for PD-1 and compare the function and expression of PD-L1 and PD-L2. Engagement of PD-1 by PD-L2 dramatically inhibits T cell receptor (TCR)-mediated proliferation and cytokine production by CD4+ T cells. At low antigen concentrations, PD-L2-PD-1 interactions inhibit strong B7-CD28 signals. In contrast, at high antigen concentrations, PD-L2-PD-1 interactions reduce cytokine production but do not inhibit T cell proliferation. PD-L-PD-1 interactions lead to cell cycle arrest in G0/G1 but do not increase cell death. In addition, ligation of PD-1 + TCR leads to rapid phosphorylation of SHP-2, as compared to TCR ligation alone. PD-L expression was up-regulated on antigen-presenting cells by interferon gamma treatment and was also present on some normal tissues and tumor cell lines. Taken together, these studies show overlapping functions of PD-L1 and PD-L2 and indicate a key role for the PD-L-PD-1 pathway in regulatingT cell responses.
CD150 (SLAM/IPO-3) is a cell surface receptor that, like the B cell receptor, CD40, and CD95, can transmit positive or negative signals. CD150 can associate with the SH2-containing inositol phosphatase (SHIP), the SH2-containing protein tyrosine phosphatase (SHP-2), and the adaptor protein SH2 domain protein 1A (SH2D1A/DSHP/SAP, also called Duncan's disease SH2-protein (DSHP) or SLAM-associated protein (SAP)). Mutations in SH2D1A are found in X-linked lymphoproliferative syndrome and non-Hodgkin's lymphomas. Here we report that SH2D1A is expressed in tonsillar B cells and in some B lymphoblastoid cell lines, where CD150 coprecipitates with SH2D1A and SHIP. However, in SH2D1A-negative B cell lines, including B cell lines from X-linked lymphoproliferative syndrome patients, CD150 associates only with SHP-2. SH2D1A protein levels are up-regulated by CD40 cross-linking and down-regulated by B cell receptor ligation. Using GST-fusion proteins with single replacements of tyrosine at Y269F, Y281F, Y307F, or Y327F in the CD150 cytoplasmic tail, we found that the same phosphorylated Y281 and Y327 are essential for both SHP-2 and SHIP binding. The presence of SH2D1A facilitates binding of SHIP to CD150. Apparently, SH2D1A may function as a regulator of alternative interactions of CD150 with SHP-2 or SHIP via a novel TxYxxV/I motif (immunoreceptor tyrosine-based switch motif (ITSM)). Multiple sequence alignments revealed the presence of this TxYxxV/I motif not only in CD2 subfamily members but also in the cytoplasmic domains of the members of the SHP-2 substrate 1, sialic acid-binding Ig-like lectin, carcinoembryonic Ag, and leukocyte-inhibitory receptor families.
During activation, T cells express receptors for receiving positive and negative costimulatory signals. Here we identify the B and T lymphocyte attenuator (BTLA), an immunoglobulin domain-containing glycoprotein with two immunoreceptor tyrosine-based inhibitory motifs. BTLA is not expressed by naive T cells, but it is induced during activation and remains expressed on T helper type 1 (T(H)1) but not T(H)2 cells. Crosslinking BTLA with antigen receptors induces its tyrosine phosphorylation and association with the Src homology domain 2 (SH2)-containing protein tyrosine phosphatases SHP-1 and SHP-2, and attenuates production of interleukin 2 (IL-2). BTLA-deficient T cells show increased proliferation, and BTLA-deficient mice have increased specific antibody responses and enhanced sensitivity to experimental autoimmune encephalomyelitis. B7x, a peripheral homolog of B7, is a ligand of BTLA. Thus, BTLA is a third inhibitory receptor on T lymphocytes with similarities to cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) and programmed death 1 (PD-1).
T helper type 1 (T(H)1) immune responses are central in cell-mediated immunity, and a T(H)1-specific cell surface molecule called Tim-3 (T cell immunoglobulin domain, mucin domain) has been identified. Here we report the identification of a secreted form of Tim-3 that contains only the immunoglobulin (Ig) variable (V) domain of the full-length molecule. Fusion proteins (Tim-3-Ig) of both Tim-3 isoforms specifically bound CD4(+) T cells, indicating that a Tim-3 ligand is expressed on CD4(+) T cells. Administration of Tim-3-Ig to immunized mice caused hyperproliferation of T(H)1 cells and T(H)1 cytokine release. Tim-3-Ig also abrogated tolerance induction in T(H)1 cells, and Tim-3-deficient mice were refractory to the induction of high-dose tolerance. These data indicate that interaction of Tim-3 with Tim-3 ligand may serve to inhibit effector T(H)1 cells during a normal immune response and may be crucial for the induction of peripheral tolerance.
Although T helper (T(H)) cell-mediated immunity is required to effectively eliminate pathogens, unrestrained T(H) activity also contributes to tissue injury in many inflammatory and autoimmune diseases. We report here that the T(H) type 1 (T(H)1)-specific Tim-3 (T cell immunoglobulin domain, mucin domain) protein functions to inhibit aggressive T(H)1-mediated auto- and alloimmune responses. Tim-3 pathway blockade accelerated diabetes in nonobese diabetic mice and prevented acquisition of transplantation tolerance induced by costimulation blockade. These effects were mediated, at least in part, by dampening of the antigen-specific immunosuppressive function of CD4(+)CD25(+) regulatory T cell populations. Our data indicate that the Tim-3 pathway provides an important mechanism to down-regulate T(H)1-dependent immune responses and to facilitate the development of immunological tolerance.
Positive selection of developing thymocytes is associated with changes in cell function, at least in part caused by alterations in expression of cell surface proteins. Surprisingly, however, few such proteins have been identified. We have analyzed the pattern of gene expression during the early stages of murine thymocyte differentiation. These studies led to identification of a cell surface protein that is a useful marker of positive selection and is a likely regulator of mature lymphocyte and APC function. The protein is a member of the Ig superfamily and contains conserved tyrosine-based signaling motifs. The gene encoding this protein was independently isolated recently and termed B and T lymphocyte attenuator (Btla). We describe in this study anti-BTLA mAbs that demonstrate that the protein is expressed in the bone marrow and thymus on developing B and T cells, respectively. BTLA is also expressed by all mature lymphocytes, splenic macrophages, and mature, but not immature bone marrow-derived dendritic cells. Although mice deficient in BTLA do not show lymphocyte developmental defects, T cells from these animals are hyperresponsive to anti-CD3 Ab stimulation. Conversely, anti-BTLA Ab can inhibit T cell activation. These results implicate BTLA as a negative regulator of the activation and/or function of various hemopoietic cell types.
To study the cis- and trans-acting factors that mediate programmed death 1 (PD-1) signaling in primary human CD4 T cells, we constructed a chimeric molecule consisting of the murine CD28 extracellular domain and human PD-1 cytoplasmic tail. When introduced into CD4 T cells, this construct mimics the activity of endogenous PD-1 in terms of its ability to suppress T cell expansion and cytokine production. The cytoplasmic tail of PD-1 contains two structural motifs, an ITIM and an immunoreceptor tyrosine-based switch motif (ITSM). Mutation of the ITIM had little effect on PD-1 signaling or functional activity. In contrast, mutation of the ITSM abrogated the ability of PD-1 to block cytokine synthesis and to limit T cell expansion. Further biochemical analyses revealed that the ability of PD-1 to block T cell activation correlated with recruitment of Src homology region 2 domain-containing phosphatase-1 (SHP-1) and SHP-2, and not the adaptor Src homology 2 domain-containing molecule 1A, to the ITSM domain. In TCR-stimulated T cells, SHP-2 associated with PD-1, even in the absence of PD-1 engagement. Despite this interaction, the ability of PD-1 to block T cell activation required receptor ligation, suggesting that colocalization of PD-1 with CD3 and/or CD28 may be necessary for inhibition of T cell activation.
B and T lymphocyte attenuator (BTLA) provides an inhibitory signal to B and T cells. Previously, indirect observations suggested that B7x was a ligand for BTLA. Here we show that BTLA does not bind B7x; instead, we identify herpesvirus entry mediator (HVEM) as the unique BTLA ligand. BTLA bound the most membrane-distal cysteine-rich domain of HVEM, distinct from regions where the ligands LIGHT and lymphotoxin-alpha bound HVEM. HVEM induced BTLA tyrosine phosphorylation and association of the tyrosine phosphatase SHP-2 and repressed antigen-driven T cell proliferation, providing an example of reverse signaling to a non-tumor necrosis factor family ligand. The conservation of the BTLA-HVEM interaction between mouse and human suggests that this system is an important pathway regulating lymphocyte activation and/or homeostasis in the immune response.
Dendritic cells (DCs) include a heterogeneous family of professional APCs involved in initiation of immunity and in immunologic tolerance. Specifically, peripheral tolerance can be achieved and maintained by promoting regulatory T-cell (Treg) responses and/or T-cell anergy or deletion. Until recently, immature developmental stages of DC differentiation were believed to induce T-cell anergy or Treg cells, whereas DCs transformed into mature DCs by activation stimuli were thought to represent immunogenic DCs capable of inciting primary T-cell responses. This paradigm has been challenged by the demonstration of Treg-cell expansion by antigen-bearing, fully mature DCs. Similarly, semimature DCs with a distinctive interleukin 10 (IL-10)+ IL-12- cytokine production profile might be endowed with tolerogenic functions, supporting the concept that DC maturation per se should no longer be considered as a distinguishing feature of immunogenic as opposed to tolerogenic DCs (TDCs). Cytokine-modulated TDCs reflect an incomplete or altered status of monocyte differentiation and promote in vitro induction of Treg cells and/or in vivo protection from autoimmune diseases. Several growth factors, including IL-10, transforming growth factor beta (TGF-beta), granulocyte colony-stimulating factor (G-CSF), hepatocyte growth factor (HGF), and vasoactive intestinal peptide (VIP), modulate DC maturation and favor the differentiation of TDCs. From a therapeutic standpoint, cytokine-modulated TDCs might be beneficial for prevention and/or treatment of posttransplantation graft-versus-host disease (GVHD) and autoimmunity.
Antigen presenting cells recognize pathogens via pattern recognition receptors (PRR), which upon ligation transduce intracellular
signals that can induce innate immune responses. Because some C-type lectin-like receptors (e.g. dectin-1 and DCSIGN) were shown to act as PRR for particular microbes, we considered a similar role for dectin-2. Binding
assays using soluble dectin-2 receptors showed the extracellular domain to bind preferentially to hyphal (rather than yeast/conidial)
components of Candida albicans, Microsporum audouinii, and Trichophyton rubrum. Selective binding for hyphae was also observed using RAW macrophages expressing dectin-2, the ligation of which by hyphae
or cross-linking with dectin-2-specific antibody led to protein tyrosine phosphorylation. Because dectin-2 lacks an intracellular
signaling motif, we searched for a signal adaptor that permits it to transduce intracellular signals. First, we found that
the Fc receptor γ (FcRγ) chain can bind to dectin-2. Second, ligation of dectin-2 on RAW cells induced tyrosine phosphorylation
of FcRγ, activation of NF-κB, internalization of a surrogate ligand, and up-regulated secretion of tumor necrosis factor α
and interleukin-1 receptor antagonist. Finally, these dectin-2-induced events were blocked by PP2, an inhibitor of Src kinases
that are mediators for FcRγ chain-dependent signaling. We conclude that dectin-2 is a PRR for fungi that employs signaling
through FcRγ to induce innate immune responses.
T-cell activation is the net product of competing positive and negative signals transduced by regulatory molecules on antigen-presenting cells (APCs) binding to corresponding ligands on T cells. Having previously identified DC-HIL as a receptor expressed by APCs that contains an extracellular immunoglobulin (Ig)-like domain, we postulated that it plays a role in T-cell activation. To probe this function, we created soluble recombinant DC-HIL, which we observed to bind activated (but not resting) T cells, indicating that expression of the putative ligand on T cells is induced by activation. Binding of DC-HIL to naive T cells attenuated these cells' primary response to anti-CD3 antibody, curtailing IL-2 production, and preventing entry into the cell cycle. DC-HIL also inhibited reactivation of T cells previously activated by APCs (secondary response). By contrast, addition of soluble DC-HIL to either allogeneic or ovalbumin-specific lymphocyte reactions augmented T-cell proliferation, and its injection into mice during the elicitation (but not sensitization) phase of contact hypersensitivity exacerbated ear-swelling responses. Mutant analyses showed the inhibitory function of DC-HIL to reside in its extracellular Ig-like domain. We conclude that endogenous DC-HIL is a negative regulator of T lymphocyte activation, and that this native inhibitory function can be blocked by exogenous DC-HIL, leading to enhanced immune responses.
HGFIN, previously identified as nmb, and its homolog osteoactivin are single transmembrane proteins that are expressed in differentiated immune cells. These proteins exhibit properties that could potentiate tumorigenesis or decrease invasiveness. These seemingly opposing roles of HGFIN suggest that this protein might be central to malignancies and might also behave as a tumor suppressor. Consistent with the reported roles for HGFIN is the fact that this gene is regulated by p53 through multiple binding sites in the 5' flanking region, and is expressed in osteoblasts.
This study used siRNA to knock-out HGFIN in non-tumorigenic breast cells and ectopically expressed HGFIN in breast cancer cells. In addition, in situ hybridization studies analyzed primary breast tissues from archived breast surgeries. Reporter gene assays studied the untranslated exon 1 of HGFIN.
HGFIN expression led to reduced cell growth of breast cancer cells and reduced migration. At the molecular level, reporter gene analyses determined the untranslated exon 1 to be a negative regulator of the upstream enhancing effect. Ectopic expression of wild-type p53 in breast cancer cells that expressed endogenous mutant p53 resulted in increased HGFIN reporter gene activities.
As the majority of cancer cells have mutations in p53, further studies on the relationship between p53 and HGFIN expression, and its role in tumor genesis and bone invasion, might uncover novel therapy targets for breast and other cancers. The results show a central role for p53 in HGFIN expression, which appears to determine the behavior of the cancer cells.
Receptor-ligand interactions between APCs and T cells determine whether stimulation of the latter leads to activation or inhibition. Previously, we showed that dendritic cell-associated heparin sulfate proteoglycan-dependent integrin ligand (DC-HIL) on APC can inhibit T cell activation by binding an unknown ligand expressed on activated T cells. Because DC-HIL binds heparin/heparan sulfate and heparin blocks the inhibitory function of DC-HIL, we hypothesized that a heparin/heparan sulfate proteoglycan on activated T cells is the relevant ligand. Screening assays revealed that syndecan-4 (SD-4) is the sole heparan sulfate proteoglycan immunoprecipitated by DC-HIL from extracts of activated T cells and that blocking SD-4 abrogates binding of DC-HIL to activated T cells. Moreover, cell-bound SD-4 ligated by DC-HIL or cross-linked by anti-SD-4 Ab attenuated anti-CD3 responses, whereas knocked-down SD-4 expression led to enhanced T cell response to APC. Blockade of endogenous SD-4 using specific Ab or soluble SD-4 receptor led to augmented T cell reactions to syngeneic and allogeneic stimulation in vitro and exacerbated contact hypersensitivity responses in vivo. We conclude that SD-4 is the T cell ligand through which DC-HIL mediates its negative coregulatory function.
Transforming growth factor-beta (TGF-beta) is a key player in malignant disease through its actions on host tissues and cells. Malignant cells often secrete large amounts of TGF-beta that act on nontransformed cells present in the tumor mass as well as distal cells in the host to suppress antitumor immune responses creating an environment of immune tolerance, augmenting angiogenesis, invasion and metastasis, and increasing tumor extracellular matrix deposition. Cells of the innate immune system contribute to the high concentrations of TGF-beta found in tumor masses. In addition, dendritic cell subpopulations secreting TGF-beta contribute to the generation of regulatory T cells that actively inhibit the activity of other T cells. Elevated levels of plasma TGF-beta are associated with advanced stage disease and may separate patients into prognostically high-risk populations. Anti-TGF-beta therapy could reverse the immunosuppressive effects of this cytokine on the host as well as decrease extracellular matrix formation, decrease angiogenesis, decrease osteolytic activity, and increase the sensitivity of the malignant cells to cytotoxic therapies and immunotherapies. Phase I clinical trials of an inhibitor of TGF-beta receptor type I kinase activity and a TGF-beta neutralizing antibody are under way.
CD160, a glycosylphosphatidylinositol-anchored member of the immunoglobulin superfamily, is expressed on both cytolytic lymphocytes and some unstimulated CD4+ T cells. Here we show that CD160 expression was increased after activation of human CD4+ T cells and that crosslinking CD160 with monoclonal antibody strongly inhibited CD3- and CD28-mediated activation. We found that herpesvirus entry mediator (HVEM) was a ligand of CD160 that acted as a 'bidirectional switch' for T cell activation, producing a positive or negative outcome depending on the engagement of HVEM by CD160 and known HVEM ligands such as B and T lymphocyte attenuator (BTLA) and the T lymphocyte receptor LIGHT. Inhibition of CD4+ T cell activation by HVEM-transfected cells was dependent on CD160 and BTLA; when the cysteine-rich domain 1 of HVEM was deleted, this inhibition was lost, resulting in strong T cell activation. CD160 thus serves as a negative regulator of CD4+ T cell activation through its interaction with HVEM.
The molecular mechanisms used by regulatory T cells (Treg) to inhibit the effector phase of adaptive immune responses are still elusive. In the present work, we investigated the possibility that Treg may interfere with a basic biological function of T helper cells (TH): polarization of secretory machinery for dedicated help delivery. To address this question, we visualized by confocal microscopy different parameters of activation in TH and Treg cells interacting simultaneously with individual antigen-presenting cells (APC). Our results show that, although productive TCR engagement in TH/APC conjugates was unaffected by the presence of adjacent Treg, the reorientation of TH secretory machinery toward APC was strongly inhibited. Blocking TGF-β completely reverted Treg induced inhibition of TH polarization. Our results identify a previously undescribed mechanism by which Treg inhibit effector T cells. TGF-β produced by adjacent Treg interferes with polarization of TH secretory machinery toward APC, thus affecting a crucial step of TH-mediated amplification of the immune response.
• confocal microscopy
• immunological synapse
• T cell activation
Osteoblast development is a complex process involving the expression of specific growth factors and regulatory proteins that control cell proliferation, differentiation, and maturation. In this study, we used the rat mutation, osteopetrosis (op), to examine differences in skeletal gene expression between mutant op and normal littermates. Total RNA isolated from long bone and calvaria was used as a template for mRNA differential display. One of many cDNAs that were selectively expressed in either normal or mutant bone was cloned and sequenced and found to share some homology to the human nmb and Pmel 17 genes. This novel cDNA was named osteoactivin. Osteoactivin has an open reading frame of 1716 bp that encodes a protein of 572 amino acids with a predicted molecular weight of 63.8 kD. Protein sequence analysis revealed the presence of a signal peptide and a cleavage site at position 23. The protein also has thirteen predicted N-linked glycosylation sites and a potential RGD integrin recognition site at position 556. Northern blot analysis confirmed that osteoactivin was 3- to 4-fold overexpressed in op versus normal bone. RT-PCR analysis showed that osteoactivin is most highly expressed in bone compared with any of the other non-osseous tissues examined. In situ hybridization analysis of osteoactivin in normal bone revealed that it is primarily expressed in osteoblasts actively engaged in bone matrix production and mineralization. In primary rat osteoblast cultures, osteoactivin showed a temporal pattern of expression being expressed at highest levels during the later stages of matrix maturation and mineralization and correlated with the expression of alkaline phosphatase and osteocalcin. Our findings show that osteoactivin expression in bone is osteoblast-specific and suggest that it may play an important role in osteoblast differentiation and matrix mineralization. Furthermore, osteoactivin overexpression in op mutant bone may be secondary to the uncoupling of bone resorption and formation resulting in abnormalities in osteoblast gene expression and function. J. Cell. Biochem. 84: 12–26, 2002. © 2001 Wiley-Liss, Inc.
The processing and presentation of whole irradiated Mycobacterium tuberculosis (Mtbγ) and its purified protein derivative (PPD) by the peripheral blood monocytes from healthy Bacillus Calmette Guérin (BCG)-vaccinated individuals was investigated. To study processing and presentation as events distinct from T cell recognition and proliferation, monocytes were pulsed with antigens for varying time intervals and fixed. The kinetics of presentation indicate that up to 2 h was required for effective presentation of PPD and 2–4 h for Mtbγ, and that the ability to activate T cells declined as the time interval for which pulsing occurred was increased, so that responses were abolished by 8–10 h. Prefixed monocytes could not present Mtbγ and PPD to T cells indicating that processing was an essential requisite. Lysosomotropic agents chloroquine, monensin, and leupeptin inhibited the presentation of these antigens suggesting the role of lysosomes/endosomes in processing. Furthermore, monocytes incubated with optimal concentration of antigens for different lengths of time released determinants which were still antigenic but circumvented the need for any further processing.
Addition of nonprimed syngeneic monocytes, both untreated or paraformaldehyde fixed to cells which had been pulsed and fixed, restored the responses even at the later time periods when responses were not detected. This second interaction of the monocyte with T cells was not major histocompatibility complex restricted in that the addition of monocytes from another donor was equally effective.
From a subtractive cDNA library, we isolated several cDNA clones which showed differential expression between highly and lowly metastatic human melanoma cell lines. One clone, designated nmb, showed preferential expression in the low-metastatic cell lines and was chosen for further characterization. Sequence analysis revealed that this clone represents a novel gene, encoding a putative transmembrane glycoprotein which showed the highest homology to the precursor of pMEL17, a melanocyte-specific protein. nmb RNA expression was absent in most tumor-cell lines tested and not restricted to the melanocytic lineage. Transfection of a partial nmb cDNA into a highly metastatic melanoma cell line (BLM) resulted, in 2 of 3 transfectants, in slower subcutaneous tumor growth and, in 1 of 3 transfectants, in reduction of the potential for spontaneous metastasis in nude mice.
CD28 and CTLA-4 are related glycoproteins found on T cells. Ligation of CD28 following antigen receptor engagement provides a costimulatory signal required for T cell activation. Anti-CTLA-4 antibodies were generated to examine the role of the CTLA-4 receptor on murine T cells. Expression of CTLA-4 as a homodimer is up-regulated 2-3 days following T cell activation. Anti-CTLA-4 antibodies and Fab fragments augmented T cell proliferation in an allogeneic MLR. However, when optimal costimulation and Fc cross-linking were present, anti-CTLA-4 Mabs inhibited T cell proliferation. Together, these results suggest that the MAb may obstruct the interaction of CTLA-4 with its natural ligand and block a negative signal, or directly signal T cells to down-regulate immune function.
The negative regulation of antigen receptor signal transduction is essential for the maintenance of thresholds for activation in lymphocytes. CD45 and SHP-1 are tyrosine phosphatases that are important in maintaining the proper level of tyrosine phosphorylation. Regulation of the src family of tyrosine kinases is mediated by the coordinated action of the tyrosine kinase Csk and the tyrosine phosphatase CD45. B cell receptor signaling is negatively regulated by the recruitment of SHP-1 to bind the B cell transmembrane proteins CD22 and FcgammaRIIb1. SHP-1 also functions to negatively regulate T cell receptor signaling by dephosphorylating and inactivating tyrosine kinases.
Selectins are a family of three cell adhesion molecules (L-, E-, and P-selectin) specialized in capturing leukocytes from the bloodstream to the blood vessel wall. This initial cell contact is followed by the selectin-mediated rolling of leukocytes on the endothelial cell surface. This represents the first step in a cascade of molecular interactions that lead to leukocyte extravasation, enabling the processes of lymphocyte recirculation and leukocyte migration into inflamed tissue. The central importance of the selectins in these processes has been well documented in vivo by the use of adhesion-blocking antibodies as well as by studies on selectin gene-deficient mice. This review focuses on the molecular mechanisms that regulate expression and function(s) of the selectins and their ligands. Cell-surface expression of the selectins is regulated by a variety of different mechanisms. The selectins bind to carbohydrate structures on glycoproteins, glycolipids, and proteoglycans. Glycoproteins are the most likely candidates for physiologically relevant ligands. Only a few glycoproteins are appropriately glycosylated to allow strong binding to the selectins. Recently, more knowledge about the structure and the regulated expression of some of the carbohydrates on these ligands necessary for selectin binding has been accumulated. For at least one of these ligands, the physiological function is now well established. A novel and exciting aspect is the signaling function of the selectins and their ligands. Especially in the last two years, convincing data have been published supporting the idea that selectins and glycoprotein ligands of the selectins participate in the activation of leukocyte integrins.
Two groups have now reported the viability of mice that lack syndecan-4. These mice have wound healing/angiogenesis problems, and fibroblasts from these animals differ in adhesion and migration from normal. This is consistent with recent in vitro data indicating a need for signaling via syndecan-4 for focal adhesion formation, and reports that overexpression of proteins that bind syndecan-4 can modify cell adhesion and migration.
Ultraviolet B (UVB) radiation is an important inducer of many biologic changes in skin, of which keratinocytes are a key target. To gain better insight into changes in gene expression generated in the early phase after UVB exposure, we used complementary RNA (cRNA) microarray hybridization to compare differences in mRNA expression of UVB-irradiated (single dose of 100 J/m2 broad-band UVB) and sham-irradiated primary cultured human keratinocytes. Six hours after irradiation, total RNA was isolated from keratinocytes, and cRNA was synthesized and hybridized to a GeneChip expression array (Affymetrix) consisting of 6800 genes. Based on a threshold of > twofold change, 187 genes (2.8%) were designated to be the most UVB-responsive. Surprisingly, none of these genes had been shown previously to be modulated by UVB. Conversely, several genes in the microarray that had been reported previously to be UVB- responsive by other methods showed less (< twofold) or no change. Northern blotting of seven differentially modulated genes produced results similar to those derived from microarray technology, thereby validating the accuracy of screening. Clustering based on known or likely functions indicated that among 88 upregulated genes, nine encode for cytochrome c subunits, six for ribosomal proteins, and two for regulators of apoptosis. By contrast, many of the 99 downregulated genes are involved in transcription, differentiation and transport. These findings indicate that keratinocytes respond to a single low dose of broad-band UVB irradiation by enhancing processes involved in energy production and translation, while suppressing those related to transcription, differentiation and transport.
Virtually every cell type in metazoan organisms produces heparan sulfate. These complex polysaccharides provide docking sites for numerous protein ligands and receptors involved in diverse biological processes, including growth control, signal transduction, cell adhesion, hemostasis, and lipid metabolism. The binding sites consist of relatively small tracts of variably sulfated glucosamine and uronic acid residues in specific arrangements. Their formation occurs in a tissue-specific fashion, generated by the action of a large family of enzymes involved in nucleotide sugar metabolism, polymer formation (glycosyltransferases), and chain processing (sulfotransferases and an epimerase). New insights into the specificity and organization of the biosynthetic apparatus have emerged from genetic studies of cultured cells, nematodes, fruit flies, zebrafish, rodents, and humans. This review covers recent developments in the field and provides a resource for investigators interested in the incredible diversity and specificity of this process.
Autoreactive lymphocytes are suppressed in healthy individuals by so-called peripheral tolerance. Accumulating evidence indicates that co-receptor signaling plays a pivotal role in the regulation of autoreactive lymphocytes. The positive regulatory co-receptors CD28 and inducible co-stimulator (ICOS) transduce stimulatory cosignals, whereas the negative regulatory co-stimulators CTLA-4 and PD-1 are critical for the regulation of peripheral tolerance and autoimmunity. PD-1 deficient mice develop lupus-like glomerulonephritis and arthritis on a C57Bl/6 background and autoimmune-dilated cardiomyopathy on a BALB/c background.
The B and T lymphocyte attenuator, BTLA, is a recently discovered Ig superfamily member. BTLA engagement results in downregulation of T-cell activation, and mice deficient in BTLA show increased incidence and severity of autoimmune disorders. A novel B7 family member, B7x (B7S1, B7-H4), has been proposed as the ligand for BTLA. A B7x-Ig fusion protein inhibits T-cell activation and interleukin-2 (IL-2) production. Thus, cytotoxic T lymphocyte antigen-4 (CTLA-4), programmed death-1 (PD-1) and BTLA comprise an expanding set of inhibitory receptors expressed on lymphocytes.
Antigen-presenting cells (APC) are considered to play a critical role in promoting the (re)activation of potentially autoreactive T cells in multiple sclerosis (MS), an inflammatory demyelinating disorder of the central nervous system (CNS). B7-H1 (PD-L1) is a novel member of the B7 family proteins which exert costimulatory and immune regulatory functions. Here we characterize the expression and functional activity of B7-H1 expressed on monocytes and dendritic cells (DC) of healthy donors and MS patients. B7-H1 is constitutively expressed on monocytes and differentially matured DC, but not on B cells. IFN-beta, the principle immune modulatory agent used for the treatment of MS, strongly enhances B7-H1 expression on monocytes and semi-matured DC, but not B cells, in vitro. Importantly, B7-H1 expressed on APC strongly inhibits autologous CD4 T-cell activation. Neutralization of B7-H1 on monocytes or differentially matured monocyte-derived DC markedly increases the secretion of the pro-inflammatory cytokines, IFN-gamma and IL-2, T-cell proliferation, and the expression of T-cell activation markers. B7-H1 exhibits strong inhibitory effects when expressed on monocytes, immature or semi-mature DC, but less so when expressed on fully matured DC. B7-H1-dependent immune inhibition is in part mediated by CD4/CD25+ regulatory T cells. There is no difference in the baseline expression levels of monocytic B7-H1 between untreated MS patients and healthy donors. However, both groups show a significant concentration-dependent up-regulation of B7-H1 mRNA and protein in response to IFN-beta in vitro. Serial measurements of B7-H1 mRNA in MS patients before and 6 months after initiation of IFN-beta therapy corroborated the relevance of these results in vivo: Nine of nine patients showed a significant increase in B7-H1 mRNA levels after 6 months of IFN-beta therapy (median 1.04 vs. 8.78; p<0.05, two-sided t-test). Accordingly, protein expression of B7-H1 on monocytes was up-regulated after 24 h of IFN-beta application. In summary, B7-H1 expressed on APC acts as a strong inhibitor of autologous CD4 T-cell activation and may thus contribute to the maintenance of peripheral immune tolerance. IFN-beta up-regulates B7-H1 in vitro and in MS patients in vivo and might represent a novel mechanism how IFN-beta acts as a negative modulator on APC T-cell interactions in the periphery.
Stromal cell-derived factor-1 (SDF-1)/CXCL12, the ligand for CXCR4, induces signal transduction. We previously showed that CXCL12 binds to high- and low-affinity sites expressed by primary cells and cell lines, and forms complexes with CXCR4 as expected and also with a proteoglycan, syndecan-4, but does not form complexes with syndecan-1, syndecan-2, CD44 or beta-glycan. We also demonstrated the occurrence of a CXCL12-independent heteromeric complex between CXCR4 and syndecan-4. However, our data ruled out the glycosaminoglycan-dependent binding of CXCL12 to HeLa cells facilitating the binding of this chemokine to CXCR4. Here, we demonstrate that CXCL12 directly binds to syndecan-4 in a glycosaminoglycan-dependent manner. We show that upon stimulation of HeLa cells by CXCL12, CXCR4 becomes tyrosine phosphorylated as expected, while syndecan-4 (but not syndecan-1, syndecan-2 or beta-glycan) also undergoes such tyrosine phosphorylation. Moreover, tyrosine-phosphorylated syndecan-4 from CXCL12-stimulated HeLa cells physically coassociates with tyrosine phosphorylated CXCR4. Pretreatment of the cells with heparitinases I and III prevented the tyrosine phosphorylation of syndecan-4, which suggests that the heparan sulfate-dependent binding of SDF-1 to this proteoglycan is involved. Finally, by reducing syndecan-4 expression using RNA interference or by pretreating the cells with heparitinase I and III mixture, we suggest the involvement of syndecan-4 and heparan sulfate in p44/p42 mitogen-activated protein kinase and Jun N-terminal/stress-activated protein kinase activation by action of CXCL12 on HeLa cells. However, these treatments did not modify the calcium mobilization induced by CXCL12 in these cells. Therefore, syndecan-4 behaves as a CXCL12 receptor, selectively involved in some transduction pathways induced by SDF-1, and heparan sulfate plays a role in these events.
Cell surface proteoglycans play an important part in the functional and metabolic behaviour of leucocytes. We studied the expression of cell surface proteoglycans in human monocytes, in monocyte-derived immature and mature dendritic cells and in macrophages by metabolic labelling with [(35)S]-sulphate, reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blotting. Immature dendritic cells had the highest metabolic activity for the synthesis of cell surface proteoglycans. The major part of these proteoglycans was in phosphatidylinositol-anchored form and was released after treatment with phospholipase C. A minor part was released by trypsin. Digestion with chondroitinase ABC and mild HNO(2) treatment showed that cell surface proteoglycans had a higher proportion of chondroitin sulphate, both in the phospholipase C and trypsin fractions, suggesting that at least some glypicans contained chondroitin sulphate chains. RT-PCR detected the transcripts of glypicans 1, 3, 4 and 5 and all syndecans. Immature dendritic cells expressed a most complex spectrum of glypicans and syndecans, glypican-1 and syndecan-1 being expressed preferentially by this type of cells. Mature dendritic cells expressed glypican-3, which was not present in other lineages. These results suggest that different mononuclear cells synthesize cell surface proteoglycans actively with characteristic expression of different syndecans and glypicans genes, depending on the degree of cell differentiation and/or maturation.
Hepatitis C virus (HCV) infection is a leading cause of chronic liver disease with over 200 million individuals infected worldwide. The vast majority of acutely infected humans develop chronic infection, which is characterized by attenuated antiviral T-cell responses. The mechanisms leading to such attenuation/suppression are poorly understood. It has been proposed that dysfunction of antigen-presenting cells (APC) may underlie the downregulation of antiviral immune responses. However, studies using bulk or in vitro-derived APC populations have resulted in conflicting reports. In this study, we evaluated the functional and immunophenotypic features of ex vivo-purified dendritic cell (DC) subsets during chronic HCV infection. We found that plasmacytoid DC (PDC) from HCV-infected patients (HCV-PDC) showed a striking deficit in IFN-alpha production in response to CpG stimulation. In addition, we found that myeloid DC (MDC) from these patients showed a diminished capacity to induce a mixed lymphocyte response (MLR), correlating with lower levels of HLA-DR and CD86 expression and higher IL-10 production in response to poly-IC stimulation. In contrast, HCV-PDC showed increased ability to stimulate an MLR. Of note, within the HCV-PDC preparation, we noted a distinctly expanded DC subset that expressed some markers of MDC, but showed significantly lower HLA-DR and CD86 expression, suggesting an expansion of DC at an immature/intermediate stage of differentiation. Our studies demonstrate distinct and contrasting dysfunctional features in DC subsets and underscore the importance of evaluating APC subpopulations separately.
Syndecans bind to cell adhesion molecules, growth factors and cytokines, and can act as coreceptors, and in this way modulate leukocyte cell function. Here, expression of the syndecans on primary human CD4 T cells was examined. Cell stimulation dramatically increased the amount of syndecan-4, and in a lower extent that of syndecan-2. Expression of syndecan-2 and -4 show different induction kinetics. Whereas syndecan-4 expression is fast and significant, that of syndecan-2 is more delayed and short-lived decreasing its mRNA expression at day 4. Both CD45RA+ naive and CD45RA- memory CD4 T cells express syndecan-2 and -4 upon activation. When incubated with human peripheral blood lymphocytes in a mixed leukocyte reaction, anti-syndecan-4 but not anti-syndecan-2 antibodies, decreased T cell proliferation. However, cross-linking of cell-bound syndecan-2 or syndecan-4 via immobilized antibodies blocked proliferation and decreased TNF production of T cells in the presence of optimal levels of anti-CD3. These findings suggest that syndecan-2 and -4 act as inhibitors of T cell activation. We also investigated the role that MAPK signalling pathways play in control of syndecan expression in T cells. We show that production of syndecan-2 but not syndecan-4 requires signaling via p38 MAP kinase alpha/beta in T CD4 cells. As mechanisms that confer syndecan-2 expression are unknown, we analyse the chromatin hypersensitivity of syndecan-2 promoter proximal region in Jurkat T cells and endothelial cells. The analysis reveals a chromatin accessible site in the +3.5kb intronic region, concomitant with a region showing high evolutionary conservation. We isolate and analyse 5'-flanking regions of human syndecan-2 gene, by transfection assays. The +3.5kb hypersensitive site in the intronic region demonstrates basal promoter activity in Jurkat. This study provides evidence for the up-regulation of syndecan-2 and -4 in human primary CD4 T cells during in vitro activation and suggest an inhibitory role for these syndecans in CD4 T cells.
Selective gene silencing by RNA interference (RNAi) has been shown to be an efficient method for the targeted manipulation of cellular functions. In this study we describe for the first time electroporation as a suitable and efficient method for the delivery of small interfering RNA (siRNA) into murine bone marrow-derived dendritic cells (BM-DC). Using a fluorescein-labeled non-silencing siRNA duplex, we established an electroporation protocol yielding routinely >90% positive cells. We investigated the effects of siRNA electroporation on BM-DC viability, phenotype and ability to induce allogeneic T cell proliferation. Finally, using siRNAs directed against MAPK1 and the transcription factor HIF-1alpha we were able to demonstrate an efficient knock down of cellular mRNA- and protein level in electroporated BM-DC. Furthermore, knocking down the transcription factor HIF-1alpha impeded hypoxic induction of HIF-1alpha target genes. We therefore propose siRNA electroporation into murine BM-DC as an efficient method to manipulate BM-DC function without the use of chemical transfection reagents. This new approach is superior to lipofection regarding detrimental effects of lipid-based transfection agents on BM-DC immunobiology.
Syndecan-4 and focal adhesion function
- Woods
Woods A, Couchman JR. Syndecan-4 and focal adhesion function. Curr. Opin. Cell Biol. 2001;
13:578–583. [PubMed: 11544026]
CD 160 inhibits activation of human CD4+ T cells through interaction with herpesvirus entry mediator
- Cai