Carcinoembryonic Antigen-related Cellular Adhesion Molecule Research Articles

Monoclonal antibody targeting CEACAM1 enhanced the response to anti-PD1 immunotherapy in non-small cell lung cancer

Carcinoembryonic antigen-related cellular adhesion molecule 1 (CEACAM1), an extensively studied cell surface molecule, mainly expressed by certain epithelial, endothelial, lymphoid and myeloid cells, and is an attractive target for cancer immunotherapy. Here, to investigate the anti-tumor effects and mechanisms of CEACAM1 antibody, we prepared the antibody and explored its anti-tumor effects on Non-small Cell Lung Cancer (NSCLC) in vitro and in vivo. Firstly, antigen of human CEACAM1 recombinant protein was immunized on BALB/c mice and the high-affinity mouse anti-human monoclonal antibody 3C11 was selected by hybridoma technique. Next, ELISA was applied to detect the blocking effects of 3C11 on CEACAM1-CEACAM1 and CEACAM1-CEACAM5. Then, cell assays and ELISA were used to evaluate the role of 3C11 in blocking CEACAM1-CEACAM1 immunosuppressive signal transduction between dendritic cells (DCs) and T cells or natural killer cells (NK) and tumor cells. Finally, the synergistic anti-tumor effect of 3C11 combined with anti-PD-1 antibody was evaluated through cell stimulation assays and NCI-H358-induced tumor models in mice. The results showed the EC50 of 3C11 binding to NCI-H358 or exhausted T cells were 0.04971 μg/mL and 0.03475 μg/mL, respectively. 3C11 activated the exhausted T cells and enhanced the killing effect of NK by blocking CEACAM1-CEACAM1. In addition, the combination of 3C11 and anti-PD1 antibody produced synergistic anti-tumor effect on NSCLC. Its improved tumor growth inhibition value (TGI) of anti-PD-1 from 18 % to 85 % in vivo. These findings suggest that 3C11 can be considered an effective immunotherapy drug for NSCLC.

Correlated STORM-homoFRET imaging reveals highly heterogeneous membrane receptor structures

Mapping the self-organization and spatial distribution of membrane proteins is key to understanding their function. Developing methods that can provide insight into correlations between membrane protein colocalization and interactions is challenging. We report here on a correlated stochastic optical reconstruction microscopy/homoFRET imaging approach for resolving the nanoscale distribution and oligomeric state of membrane proteins. Using live cell homoFRET imaging of carcinoembryonic antigen-related cellular adhesion molecule 1, a cell-surface receptor known to exist in a complex equilibrium between monomer and dimer/oligomer states, we revealed highly heterogeneous diffraction-limited structures on the surface of HeLa cells. Furthermore, correlated super-resolved stochastic optical reconstruction microscopy imaging showed that these structures comprised a complex mixture and spatial distribution of self-associated carcinoembryonic antigen-related cellular adhesion molecule 1 molecules. In conclusion, this correlated approach provides a compelling strategy for addressing challenging questions about the interplay between membrane protein concentration, distribution, interaction, clustering, and function.

P010 Adherent-Invasive Escherichia Coli strain O83:H1 induces inflammatory response in Crohn’s disease

Abstract Background Several studies suggest that Adherent-Invasive Escherichia Coli (AIEC) colonize the ileal mucosa of patients with Crohn’s disease (CD). This strain is able to adhere to and invade intestinal epithelial cells, and survive within macrophages, prompting an inflammatory response. The aim of this study was to investigate, by using an organ culture model, the ability of AIEC strain O83:H1 to colonize intestinal epithelial cells of CD patients, inducing chronic inflammation. Methods An organ culture model conducted with colonic biopsies derived from CD patients was set up to evaluate the ability of AIEC strain 083:H1, compared to a non-pathogenic (NP) E. Coli strain, to upregulate CEACAM 6 (CarcinoEmbryonic Antigen-related Cellular Adhesion Molecule 6), LAMP-1 (Lysosome Associated Membrane Protein 1), ICAM-1 (Intracellular Adhesion Molecule) and HLA-DR antigen expression, by immunohistochemistry, and to induce IFN-γ, TNF-α and IL-8 production, by RT-qPCR. The staining of epithelial cells that expressed CEACAM6 and LAMP1 as well as the expression of ICAM1 on blood vessels was evaluated in terms of staining intensity. The number of LAMP1 and HLA-DR lamina propria mononuclear cells (LPMNC) was evaluated within a total area of 1 mm2 of lamina propria. Results Expression of CEACAM6 on intestinal epithelial cells, and the expression of LAMP-1 either on epithelium as well as in the LPMNCs, were significantly increased (P<0,05) in the biopsies cultured with the AIEC strain O83:H1 compared to the biopsies cultured with NP strain. ICAM-1 and HLA-DR were significantly increased (P<0,05) on blood vessels and on LPMNCs, respectively, in presence of AIEC strain O83:H1 as compared with NP strain. Moreover we observed a higher level of IFN-γ, TNF-α and IL-8 mRNA trascripts in biopsies cultured with AIEC strain 083:H1 than in biopsies cultured with NP strain. Conclusion Our data suggest that AIEC strain O83:H1 in CD is able to adhere to and invade the intestinal epithelial cells, and to play an important role in activating chronic inflammatory responses observed in CD patients.

Expression and diagnostic value of macrophage inflammatory protein-3α in patients with hepatitis B-related acute-on-chronic liver failure

Objective: To screen serum protein markers and evaluate their diagnostic application value in hepatitis B-related acute-on-chronic liver failure (HBV-ACLF). Methods: Serum samples of patients with HBV-ACLF, chronic hepatitis B (CHB) and normal healthy volunteers (n = 5/group) were determined by cytokine antibody chip in line with the Chinese Diagnostic Standards Study for HBV-ACLF (COSSH-ACLF) cohort. The differentially expressed proteins significance were identified by microarray analysis and prediction. The preliminary serological markers of HBV-ACLF were screened for diagnosis. The potential markers were determined by enzyme-linked immunosorbent assay (ELISA), area under the receiver operating characteristic curve (AUROC) analysis and liver tissue immunohistochemistry for the diagnosis of HBV-ACLF. Student t-test or Mann-Whitney U test were used to compare the continuous measurement data between the two groups, and analysis of variance and Kruskal-Wallis test were used to compare the continuous measurement data between multiple groups. Results: Cytokine antibody chip preliminary screening results showed that the expression levels of these six cytokines, namely, macrophage inflammatory protein 3α (MIP-3α), hepatocyte growth factor, E-selectin, osteopontin, growth differentiation factor 15 and carcinoembryonic antigen-related cellular adhesion molecule 1 were significantly increased in the HBV-ACLF group. Among them, the expression level of MIP-3α was significantly higher in the HBV-ACLF group (99.6 times higher than CHB group and 146.9 times higher than healthy volunteers' group, respectively, P < 0.0001) as validated by serum ELISA in 132 HBV-ACLF cases, 91 CHB cases and 72 healthy volunteers. AUROC analysis showed that the high expression of MIP-3α could be used as a marker to distinguish patients with HBV-ACLF from CHB. The AUROC was 0.995 (95% CI: 0.990 ~ 1.000), with sensitivity and specificity of 95.5% and. 98.9%, respectively. Immunohistochemistry showed that MIP-3α was positively expressed in HBV-ACLF-derived liver tissues, and negatively expressed in CHB-derived liver and normal liver tissues. Conclusion: Serum MIP-3α level is closely related to the pathological characteristics of HBV-ACLF. Therefore, it may be used as a potential serological marker for the diagnosis of HBV-ACLF.

Effect of Carcinoembryonic Antigen-related Cell Adhesion Molecule-binding Recombinant Polypeptide on the Killing of Neisseria meningitidis by Human Neutrophils

Background: Neutrophils are an essential part of innate immunity and play a crucial role in controlling infection caused by Neisseria meningitidis. The Moraxella catarrhalis ubiquitous surface protein A1 (UspA1)-based recombinant polypeptide binds to the carcinoembryonic antigen-related cellular adhesion molecule (CEACAM) 1 receptor on host cells and blocks binding of the mucosal pathogens to human epithelial cells and T cells. Aim of the study: Since the CEACAM1 receptor is expressed on neutrophils, the aim of this study was to investigate the effect of CEACAM1-binding recombinant polypeptide on the ability of neutrophils to kill Neisseria meningitidis. Methods: The effect of CEACAM1-binding recombinant polypeptide on the phagocytic killing of Neisseria meningitidis by neutrophils was assessed by incubation of neutrophils with CEACAM1-binding recombinant polypeptide (UspA1 527–665) or with CEACAM1-non-binding polypeptide control (UspA1 659–863) for one hour before infection with Neisseria meningitidis. The surviving bacteria were released and counted. Results: 30 minutes after infection of neutrophils with Neisseria meningitidis, the survival of bacteria in presence of CEACAM1-binding recombinant polypeptide was 64% compared to 52% with control peptide and 43% without peptide. However, one-hour after infection, the surviving bacteria was 32% in presence of CEACAM1-binding recombinant polypeptide compared to 18% with control peptide and 22% without peptides. Conclusion: Although CEACAM1-binding polypeptide reduced the killing of Neisseria meningitidis by neutrophils, it did not entirely stop phagocytosis and killing of bacteria.

CEACAM1 Inhibited IκB-α/NF-κB Signal Pathway Via Targeting MMP-9/TIMP-1 Axis in Diabetic Atherosclerosis.

Atherosclerosis (AS) is the most common and serious complication in type 2 diabetes mellitus (T2DM). Recent studies have emphasized that inflammation is the main cause of atherosclerosis. Studies have shown that carcinoembryonic antigen-related cellular adhesion molecule 1 (CEACAM1) regulates the expression of matrix metallopeptidase 9 (MMP-9) after ischemic stroke to reduce inflammation. The aim of this study was to elucidate potential molecular mechanism of CEACAM1 on the inflammatory response in atherosclerosis. The serum levels of CEACAM1, MMP-9, and tissue inhibitors of metalloproteinase 1 (TIMP-1) in T2DM patients and healthy control was detected. The results showed that the levels of CEACAM1 and TIMP-1 were significantly decreased, and the levels of MMP-9 were significantly higher than those in the control group. Moreover, we also observed the effect of CEACAM1 on atherosclerosis in T2DM rats. Hematoxylin & eosin (HE) staining and oil red staining showed that CEACAM1 recombinant protein reduced intima-media thickness and the area of atherosclerotic plaques. To further explore the molecular mechanism of CEACAM1 regulating MMP-9/TIMP-1, we conducted experiments in rat aorta vascular endothelial cells and rat aorta smooth muscle cells. The result showed that CEACAM1 inhibits inflammatory response via MMP-9/TIMP-1 axis. Taken together, CEACAM1 attenuates diabetic atherosclerosis by inhibition of IκB/NF-κB signal pathway via MMP-9/TIMP-1 axis, which indicate that CEACAM1 is potentially amenable to therapeutic manipulation for clinical application in atherosclerosis in T2DM.

Functional and Structural Studies of Opa Proteins from Neisseria

Neisseria gonorrhoeae (Gc) and Neisseria meninigitidis are pathogenic bacteria causing the sexually transmitted infection gonorrhea and meningococcal meningitis, respectively. Opacity-associated (Opa) proteins in Neisseria trigger engulfment of the bacteria into human cells via their interaction with members of the carcinoembryonic antigen-related cellular adhesion molecule (CEACAM) family. The structure of an Opa from Gc was determined but in vitro binding and selectivity between Opa and CEACAM have not recapitulated in vivo and in situ findings. There is evidence that lipooligosaccharide (LOS), the dominant component of the neisserial outer membrane, can bind Opa proteins on adjacent bacteria, and that changes in LOS structure can affect Opa expression, Opa-CEACAM interactions, and Neisseria survival. For these reasons, investigations of 1) the impact of LOS on the structure and dynamics of Opa loops, as well as 2) Opa-CEACAM binding affinity and selectivity in the presence of LOS are underway. Progress towards understanding the interaction between LOS and Opa will be presented.

Mapping Ceacam 1-4L's Nanoscale Spatial Distribution, Structure, Dynamics, and Association with Lipid Ordered Domains by Super-Resolution Microscopy

Super-resolution microscopy has provided novel quantitative insights into the nanoscale spatial distribution of membrane proteins, including the observation that some systems form micro- and nano-sized clusters. However, details of the protein's self-association state (monomer / dimer / oligomer) within the different cluster types are often missing. Since it has been shown that a protein's oligomeric form might be a crucial determinant of its function, it is important to have a technique capable of measuring both spatial distribution and association state. To that end, we have applied a correlated dSTORM/homo-FRET approach to characterize CEACAM 1-4L, the progenitor isoform of the carcinoembryonic antigen-related cellular adhesion molecule (CEACAMs) family. CEACAMs are cell surface glycoproteins involved in homo- and hetero-philic intercellular interactions that control cellular growth, differentiation, tumourigenesis, inflammation and infection. While CEACAM1-4L is known to exist as a monomer or oligomer with the former being hypothesized to be involved in activatory intercellular interactions, the basis for its spatial distribution and association state as a function of distribution remain poorly understood. Our correlated approach involved live cell homo-FRET studies of eYFP-CEACAM1-4L in HeLa cells followed by fixed cell dSTORM imaging performed using nanobody labeling with Alexa Fluor 647 (AF647) conjugated to an anti-eYFP single domain antibody. Cluster analysis of the dSTORM datasets using Voronoi tessellation revealed that the CEACAM1-4L nanoclusters were preferentially comprised of monomeric CEACAM1-4L while microclusters contained a heterogeneous distribution of CEACAM1-4L monomers and oligomers. To further characterize these clusters, super-resolution imaging using Alexa-647 labelled cholera toxin was performed to examine the co-localization of lipid ordered domains, which are known to drive membrane protein lateral distribution and clustering, with CEACAM1-4L. Finally, live-cell single particle tracking (SPT) of CEACAM1-4L was performed to investigate the putative confined, obstructed, free diffusion, and directed motion dynamics of CEACAM1-4L.

Characterization of CEACAM1 and Lipid Raft Nanoclustering, Association and Structure by dSTORM and homo-FRET Imaging

The advent of super-resolution microscopy has revealed that most membrane proteins cluster at nanoscale lengths. Clustering and compartmentalization of membrane proteins is partially caused by lipid rafts that exist at small spatial (10-100 nm) and temporal scales, that are not perceptible using classic fluorescence microscopy (1,2). Carcinoembryonic antigen-related cellular adhesion molecules (CEACAMs) are cell surface glycoproteins involved in homo- and hetero-philic intercellular interactions that control cellular growth, differentiation, tumourigenesis, inflammation and infection. Here, we investigate the association of lipid rafts with CEACAMs using direct stochastic optical reconstruction microscopy (dSTORM). We have previously demonstrated using live cell TIRF-homoFRET-dSTORM that CEACAM1 can exist as monomers and oligomers at the cell membrane, with the monomers being predominantly organized in clustered regions and the dimers in more diffuse regions. We super-resolved CEACAM1 and lipid rafts using an anti-GFP labeled nanobody and a labeled Cholera Toxin subunit B, respectively. This approach allows us to obtain super-resolved images from which coordinates with a localization precision of 20 nm can be derived. Subsequent clustering analyses allowed us to quantify the degree of association between lipid rafts and CEACAM1. Moreover, correlating homo-FRET CEACAM data with these super-resolved maps allowed us to elucidate CEACAM monomer/oligomer distribution and association with lipid rafts. Our results show that both CEACAM1 and lipid rafts exhibit nano and micro-sized clusters of various shapes and partial spatial colocalization. This reinforces the body of work suggesting that lipid rafts are necessary to support cell adhesion, pathogen binding and intercellular signalling.

Neisserial Opa Protein Loop Dynamics and Mechanism of Interaction with Host CEACAM Receptors

Neisseria gonorrhoeae and N. meningitides are human pathogens which infect millions each year and are becoming increasingly antibiotic resistant. These bacteria utilize opacity-associated (Opa) proteins to mediate bacterial uptake into non-phagocytic and phagocytic cells as a means of infection. Opa proteins primarily engage carcinoembryonic antigen-related cellular adhesion molecules (CEACAMs), hijacking host cellular mechanisms to induce bacterial engulfment. The Opa family of proteins are outer membrane β-barrels with eight transmembrane strands and four extracellular loops. Opa proteins bind CEACAMs with high affinity and specificity, yet the loops important to receptor engagement are highly diverse in sequence among Opa variants. We aim to determine how sequence diversity in the receptor-interacting domains of Opa loops facilitates engagement of CEACAM receptors. Results from CW-EPR and NMR relaxation experiments indicate the Opa loops are dynamic on the nanosecond timescale in lipid and detergent environments. Such flexibility may enable sampling of transient structures and interactions that promote receptor engagement. To determine if the conformation of the bound loops is sampled, the structure of the Opa-CEACAM complex is needed. Towards this end, a model of the Opa60-CEACAM1 binding complex will be determined using a hybrid Double Electron Electron Resonance (DEER) and molecular dynamics (MD) approach. DEER distances between Opa60 and CEACAM1 provide spatial restraints for MD simulations of the complex, and preliminary models will be presented. Elucidating the mechanism of Opa-receptor interactions will augment our understanding of the determinants promoting the entry of a foreign body into non-phagocytic cells.

Neisserial Opa Protein Dynamics and Interaction with Host Ceacam Receptors

Human pathogens Neisseria gonorrhoeae and N. meningitides are unique in their utilization of opacity-associated (Opa) proteins to mediate bacterial uptake into non-phagocytic cells. Opa proteins engage either heparan sulfate proteoglycan (HSPG) receptors or carcinoembryonic antigen-related cellular adhesion molecules (CEACAMs) to hijack host cellular mechanisms, which induces bacterial engulfment. The Opa family of proteins are eight stranded β-barrels with four extracellular loops. Regions in loops two and three contain hypervariable sequences among Opa variants and dictate receptor specificity. We aim to investigate the structural determinants of Opa-receptor interactions. Overall loop dynamics of Opa60, a CEACAM-binding Opa variant, were determined using CW-EPR and combined with the limited NMR relaxation data. Results indicate that the loops and hypervariable regions are highly mobile on the nanosecond timescale. Initial DEER experiments measured distances between Opa60 and CEACAM in the complex and preliminary models consistent with these distances will be presented. Determining the interactions between Opa and CEACAM will provide an understanding of the molecular interactions that mediate the entry of a foreign body into non-phagocytic cells.

Characterization of CEACAM1 Structure and Dynamics by Correlated homo-FRET and dSTORM Imaging

Carcinoembryonic antigen-related cellular adhesion molecules (CEACAMs) are involved in intercellular interactions that control cellular growth, differentiation, transformation, tumourigenesis, inflammation and infection. Previous studies of the CEACAM family have provided compelling evidence for the existence of a complex equilibrium of monomers and dimers (or oligomers) and have identified key factors that appear to influence the equilibrium. To date, how CEACAMs behave, interact, organize and self-associate at the cell membrane remains poorly understood. We have previously demonstrated using live cell TIRF-homoFRET imaging that a heterogeneous population of CEACAM1 monomers and oligomers exist throughout the cell membrane but that the population was skewed towards the monomeric form in actin-rich domains. However these homo-FRET maps are diffraction-limited and do not represent the true spatial distribution or dynamics of CEACAM1. In order to address these questions, we have applied direct stochastic optical reconstruction microscopy (dSTORM), to directly map the structure, distribution and oligomeric states of CEACAM1. We also applied two colour dSTORM imaging of actin filaments and intracellular CEACAM1 in order to further characterize the oligomeric form of CEACAM1 during transport from the cytosol to the cell membrane. These powerful approaches will enable us to elucidate the spatial distribution of CEACAM1, and aid in identifying correlations between CEACAM1 self-association, molecular structure, and function.

Tracking Ceacam1 Interactions and Dynamics with homo-FRET and Image Correlation Techniques

The carcinoembryonic antigen (CEA) family plays an important role in numerous normal and pathogenic processes related to cellular growth and differentiation. Crucial roles of various CEA-family members are well established while the effect of carcinoembryonic antigen-related cellular adhesion molecule 1 (CEACAM1) on immune-cell function has only recently been recognized. CEACAM1 is known to exist in both monomeric and dimeric states that are heterogeneously distributed at the cell surface. However, which form participates in cell-cell interactions and its related dynamics remain unknown. We report here details of CEACAM1's spatial distribution, oligomeric state, diffusional characteristics and dynamics as determined by live cell homo-fluorescence energy resonance transfer (homo-FRET) microscopy and imaging total internal reflection fluorescence correlation spectroscopy (ITIR-FCS).

Expression profiling of CEACAM6 associated with the tumorigenesis and progression in gastric adenocarcinoma.

Carcinoembryonic antigen-related cellular adhesion molecule 6 (CEACAM6) is a member of the immunoglobulin superfamily and has been recently reported to affect the neoplastic, metastatic, and invasive ability of malignant cells by regulating intracellular signaling pathways during tumorigenesis and progression. We investigated the expression and amplification of CEACAM6 in relation to the clinicopathological and biological significance of gastric adenocarcinoma. Expression of CEACAM6 mRNA in 75 primary gastric adenocarcinom and 20 adjacent tissues compared to normal gastric mucosas were explored using real-time quantitative-polymerase chain reaction. Immunohistochemical assays were conducted to evaluate the expression and tissue distribution of CEACAM6 protein. Overexpression of CEACAM6 mRNA in both gastric adenocarcinoma (2.513 ± 0.869) and adjacent tissues (1.171 ± 0.428) was significantly higher than the relative expressions in non-neoplastic specimens (0.594 ± 0.513) (P < 0.01). CEACAM6 protein was present in 52 (69.33%) gastric adenocarcinomas, but not in normal gastric tissues. Adenocarcinomas with elevated CEACAM6 expression were significantly associated with lymph node metastases and advanced stages. There were no relationships between CEACAM6 expression and tumor size, histological differentiation, or different subtypes, respectively. Moreover, higher expression of CEACAM6 was found to be correlated with short postoperative survival time of patients with gastric cancer. Amplification and upregulation of CEACAM6 expression was observed in human gastric adenocarcinomas, which may be correlated with the generation or transformation of malignant cells, tumor aggressive progression, and clinical outcome. CEACAM6 may be a valuable biomarker screening for gastric tumor and novel predictor for patients in advanced stages of gastric cancer.

Association of Neisseria gonorrhoeae OpaCEA with Dendritic Cells Suppresses Their Ability to Elicit an HIV-1-Specific T Cell Memory Response

Infection with Neisseria gonorrhoeae (N. gonorrhoeae) can trigger an intense local inflammatory response at the site of infection, yet there is little specific immune response or development of immune memory. Gonococcal surface epitopes are known to undergo antigenic variation; however, this is unlikely to explain the weak immune response to infection since individuals can be re-infected by the same serotype. Previous studies have demonstrated that the colony opacity-associated (Opa) proteins on the N. gonorrhoeae surface can bind human carcinoembryonic antigen-related cellular adhesion molecule 1 (CEACAM1) on CD4+ T cells to suppress T cell activation and proliferation. Interesting in this regard, N. gonorrhoeae infection is associated with impaired HIV-1 (human immunodeficiency virus type 1)-specific cytotoxic T-lymphocyte (CTL) responses and with transient increases in plasma viremia in HIV-1-infected patients, suggesting that N. gonorrhoeae may also subvert immune responses to co-pathogens. Since dendritic cells (DCs) are professional antigen presenting cells (APCs) that play a key role in the induction of an adaptive immune response, we investigated the effects of N. gonorrhoeae Opa proteins on human DC activation and function. While morphological changes reminiscent of DC maturation were evident upon N. gonorrhoeae infection, we observed a marked downregulation of DC maturation marker CD83 when the gonococci expressing CEACAM1-specific OpaCEA, but not other Opa variants. Consistent with a gonococcal-induced defect in maturation, OpaCEA binding to CEACAM1 reduced the DCs’ capacity to stimulate an allogeneic T cell proliferative response. Moreover, OpaCEA-expressing N. gonorrhoeae showed the potential to impair DC-dependent development of specific adaptive immunity, since infection with OpaCEA-positive gonococci suppressed the ability of DCs to stimulate HIV-1-specific memory CTL responses. These results reveal a novel mechanism to explain why infection of N. gonorrhoeae fails to trigger an effective specific immune response or develop immune memory, and may affect the potent synergy between gonorrhea and HIV-1 infection.

Monitoring the CEACAM1 Monomer and Dimer Distribution upon Cell-Substrate Contact

The carcinoembryonic antigen-related cellular adhesion molecule 1 (CEACAM1) is an intercellular adhesion molecule that plays a key role in processes related to cell growth, differentiation, and infection in normal and cancerous cells. Expressed on the cell surface, its extracellular domain participates in interactions with CEACAMs on neighbouring cells, as well as interacting with Opa proteins expressed on Neisseria gonorrhoeae and Neisseria meningitides in order to facilitate bacterial entry. While CEACAM1 is known to exist in both monomeric and dimeric states that are heterogeneously distributed at the cell surface, which form participates in the various inter-cellular and bacterial interactions remains a mystery. Resolving this uncertainty is fundamental to understanding the role of the receptor, as the monomer-dimer status of CEACAM1 has been shown to differentially affect its ability to bind certain downstream signalling molecules. We are using live cell fluorescence and homogeneous Forster resonance energy transfer (homo-FRET) microscopy on a combined total internal reflection fluorescence polarization microscopy (TIRFPM) and confocal microscopy platform to investigate the distribution, dynamics, and monomer-dimer equilibrium of EYFP-labeled CEACAM1. Cells expressing the labeled receptor are monitored as they come into contact with CEACAM1-engaging surfaces, such as other cells or glass dishes that have been patterned with Opa+ Neisseria gonorrhoeae. These approaches will allow us to directly examine the initial stages of CEACAM1 association with CEACAM1-interacting proteins and will answer critical questions regarding the nature of these interactions. Such insights are essential for understanding the nature of CEACAM1 signalling, function, and regulation, and ultimately targeting its functions for purposes such as cancer therapy or the treatment of infection.

Distribution of carcinoembryonic antigen‐related cellular adhesion molecules in human gingiva

Carcinoembryonic antigen-related cellular adhesion molecules (CEACAMs) are glycoproteins produced in epithelial, endothelial, lymphoid, and myeloid cells. Carcinoembryonic antigen-related cellular adhesion molecules mediate cell-cell contact and host-pathogen interactions. The aims of this study were to map the distribution and examine the regulation of CEACAMs in human gingival sites. Quantitative real-time PCR performed on human gingival biopsies from periodontitis sites revealed mRNA coding for CEACAM1, -5, -6, and -7. Immunohistochemistry showed that CEACAMs were not found in oral gingival epithelium, except for CEACAM5 in periodontitis. Carcinoembryonic antigen-related cellular adhesion molecules 1, 5, and 6 were present in the oral sulcular epithelium of periodontitis but not in that of healthy gingiva. In junctional epithelium, all three molecules were present in healthy gingiva, but in periodontitis only CEACAM1 and -6 were detected. Staining for CEACAM1 and -6 was also seen in the inflammatory cell infiltrate in periodontitis. No staining for CEACAM7 was found. Proinflammatory mediators, including lipopolysaccharide (LPS), tumour necrosis factor-α (TNF-α)/interleukin-1β (IL-1β), and interferon-γ (IFN-γ), increased the expression of CEACAM1 and CEACAM6 mRNAs in cultured human oral keratinocytes. CEACAM1 and CEACAM6 mRNAs were also strongly up-regulated upon stimulation with lysophosphatidic acid. In conclusion, the distribution of different CEACAMs was related to specific sites in the gingiva. This might reflect different functional roles in this tissue.

Defining the Roles of Human Carcinoembryonic Antigen-Related Cellular Adhesion Molecules during Neutrophil Responses to Neisseria gonorrhoeae

Symptomatic infection of humans with Neisseria gonorrhoeae is characterized by a neutrophil-rich cervical or urethral exudate, suggesting that neutrophils are important both for the clearance of these bacteria and for the pathogenesis of gonorrhea. Neisseria interacts with neutrophils through ligation of human carcinoembryonic antigen related-cellular adhesion molecules (CEACAMs) by their surface-expressed Opa proteins, resulting in bacterial binding, engulfment, and neutrophil activation. Multiple CEACAMs are expressed by human neutrophils, and yet their coexpression has precluded understanding of the relative contribution of each CEACAM to functional responses of neutrophils during neisserial infection. In this work, we directly address the role of each CEACAM during infection by introducing individual human CEACAMs into a differentiated murine MPRO cell line-derived neutrophil model. Murine neutrophils cannot bind the human-restricted Neisseria; however, we show that introducing any of the Opa-binding CEACAMs of human neutrophils (CEACAM1, CEACAM3, and CEACAM6) allows binding and entry of Neisseria into murine neutrophils. While CEACAM1- and CEACAM6-expressing neutrophils bind more bacteria, neisserial uptake via these two receptors unexpectedly proceeds without appreciable neutrophil activation. In stark contrast, neisserial engulfment via CEACAM3 recapitulates the oxidative burst and intracellular granule release seen during human neutrophil infection. Finally, by coexpressing multiple CEACAMs in our model, we show that the expression of CEACAM1 and CEACAM6 potentiate, rather than hinder, CEACAM3-dependent responses of neutrophils, exposing a cooperative role for this family of proteins during neisserial infection of neutrophils. These observations illustrate a divergence in function of CEACAMs in neutrophils and implicate the human-restricted CEACAM3 in the neutrophil innate response to neisserial infection.

The Possible Roles of OPN-Regulated CEACAM1 Expression in Promoting the Survival of Activated T Cells and the Apoptosis of Oral Keratinocytes in Oral Lichen Planus Patients

Oral lichen planus is a chronic inflammatory disorder of the oral mucosa that represents T cell-mediated autoimmune diseases. The regulation and roles of carcinoembryonic antigen-related cellular adhesion molecule 1 (CEACAM1), a novel immune molecule, in the immunopathogenesis of T cell-mediated autoimmune diseases remain unclear. In the current paper, CEACAM1 was found to be overexpressed in peripheral T cells and epithelial cells in oral lichen planus patients. A fraction of infiltrating inflammatory mononuclear cells in the lamina propria of the oral lichen planus mucosa also expressed CEACAM1. Importantly, for the first time, CEACAM1 expression in T cells and in normal human oral keratinocytes was demonstrated to be regulated differently by osteopontin in vitro. Furthermore, the apoptosis of oral keratinocytes and activated T cells can be markedly suppressed by CEACAM1-specific monoclonal antibodies. In conclusion, OPN-regulated CEACAM1 expression may play a critical role in the immunopathogenesis of oral lichen planus.

A Combined Confocal-Total Internal Reflection Fluorescence (TIRF) Single-Cell Microscopy Investigation of CEACAM1 Dynamics

The carcinoembryonic antigen-related cellular adhesion molecules (CEACAMs) represent a subset of the immunoglobulin superfamily of cell adhesion molecules that mediate intercellular adhesion. One member of this subset, CEACAM1, is particularly interesting since it is down-regulated in tumourigenic cell lines. CEACAM1 exists as an equilibrium between monomers and dimers; however, the spatial and temporal distribution upon cell-substrate contact is not known.In order to determine CEACAM1 dynamics, a coupled confocal-total internal reflection fluorescence (TIRF)-atomic force microscopy (AFM) platform was used. Live YFP-CEACAM1 labelled cells were isolated on AFM tips for controlled positioning of cells. Confocal microscopy was used to map CEACAM1 receptors over the entire cell surface. Coupled TIRF microscopy monitored the free cell surface as it was brought into contact with a substrate (i.e. another cell, a model membrane, modified glass). Confocal and TIRF microscopy homoFRET measurements were used to determine the distribution of monomeric and dimeric CEACAM1 receptors prior to and upon cell-substrate contact. Through this understanding of how molecular organization affects intercellular binding and signal transduction, it may be possible to identify peptide or pharmacological drug strategies to create CEACAM-focused therapies for cancer.View Large Image | View Hi-Res Image | Download PowerPoint Slide