Inka Scheffrahn

Carcinoembryonic Antigen-Related Cell Adhesion Molecule 1 Expression and Signaling in Human, Mouse, and Rat Leukocytes: Evidence for Replacement of the Short Cytoplasmic Domain Isoform by Glycosylphosphatidylinositol-Linked Proteins in Human Leukocytes

Carcinoembryonic Ag-related cell adhesion molecule 1 (CEACAM1), the primordial carcinoembryonic Ag gene family member, is a transmembrane cell adhesion molecule expressed in leukocytes, epithelia, and blood vessel endothelia in humans and rodents. As a result of differential splicing, CEACAM1 occurs as several isoforms, the two major ones being CEACAM1-L and CEACAM1-S, that have long (L) or short (S) cytoplasmic domains, respectively. The L:S expression ratios vary in different cells and tissues. In addition to CEACAM1, human but not rodent cells express GPI-linked CEACAM members (CEACAM5–CEACAM8). We compared the expression patterns of CEACAM1-L, CEACAM1-S, CEACAM6, and CEACAM8 in purified populations of neutrophilic granulocytes, B lymphocytes, and T lymphocytes from rats, mice, and humans. Human granulocytes expressed CEACAM1, CEACAM6, and CEACAM8, whereas human B lymphocytes and T lymphocytes expressed only CEACAM1 and CEACAM6. Whereas granulocytes, B cells, and T cells from mice and rats expressed both CEACAM1-L and CEACAM1-S in ratios of 2.2–2.9:1, CEACAM1-S expression was totally lacking in human granulocytes, B cells, and T cells. Human leukocytes only expressed the L isoforms of CEACAM1. This suggests that the GPI-linked CEACAM members have functionally replaced CEACAM1-S in human leukocytes. Support for the replacement hypothesis was obtained from experiments in which the extracellular signal-regulated kinases (Erk)1/2 were activated by anti-CEACAM Abs. Thus, Abs against CEACAM1 activated Erk1/2 in rat granulocytes, but not in human granulocytes. Erk1/2 in human granulocytes could, however, be activated by Abs against CEACAM8. We demonstrated that CEACAM1 and CEACAM8 are physically associated in human granulocytes. The CEACAM1/CEACAM8 complex in human cells might accordingly play a similar role as CEACAM1-L/CEACAM1-S dimers known to occur in rat cells.

CD30-mediated cell cycle arrest associated with induced expression of p21(CIP1/WAF1) in the anaplastic large cell lymphoma cell line Karpas 299.

One of the major characteristics of anaplastic large cell lymphomas (ALCL) is the expression of the Ki-1/CD30 antigen. While the receptor mediates NF-κB-activation in Hodgkins lymphomas, some data suggest the CD30-mediated apoptosis of other CD30-expressing cells. We were able to demonstrate that activation of CD30 leads to different effects regarding cell proliferation of the ALCL-derived cell lines Karpas 299 and JB6. Western and Northern blotting analysis revealed that CD30-induced growth inhibition of Karpas 299 cells correlated with a strong upregulation of the cell cycle inhibitor p21CIP1/WAF1. We found a non activating point mutation at codon 273 in exon 8 of the p53 gene in Karpas 299 cells which indicates an p53-independent mechanism for induced p21 expression. Abundant p21 protein expression resulted in hypophosphorylation of the retinoblastoma protein (Rb) and inhibition of the proliferating cell nuclear antigen (PCNA). CD30-stimulated cells showed no indications of apoptotic cell death, like genomic DNA fragmentation or cleavage of the caspase-3 target protein poly (ADP-ribose) polymerase (PARP). Our results indicate that CD30 is able to mediate an p21-associated cell cycle arrest in ALCL with possible implications for prognosis and clinical treatment.

CEACAM1 (CD66a) mediates delay of spontaneous and fas ligand-induced apoptosis in granulocytes

Granulocytes form the first and fastest line of defense against pathogenic infections. Their survival is limited by apoptosis, a process that is critical for the resolution of inflammation. Pro‐apoptotic and pro‐inflammatory cytokines, as well as several receptors, can alter the lifespan of granulocytes. Here we report that the carcinoembryonic antigen‐related cell adhesion molecule 1 (CEACAM1, CD66a) is involved in the regulation of granulocyte survival. Until now CEACAM1 is described to control cell proliferation, cell migration, tumor growth, angiogenesis and diverse leukocyte functions. However, very little is known about its role in granulocytes. We found that CEACAM1 expression in resting rat granulocytes is significantly higher than in other leukocyte subtypes. Stimulation led to a strongly increased CEACAM1 cell surface expression and to release of soluble CEACAM1. DNA fragmentation assays and annexin V staining revealed that binding of CEACAM1‐specific antibodies, Fab fragments and soluble CEACAM1‐Fc constructs to cell surface‐expressed CEACAM1 causes a delay of spontaneous and Fas ligand (CD95L)‐induced apoptosis. Tyrosine phosphorylation of CEACAM1‐L, its association with SHP‐1, the activation of Erk1/2 and caspase‐3 appeared to be crucial for the CEACAM1‐mediated anti‐apoptotic effect. These findings provide evidence that CEACAM1 influences the resolution of inflammation by prolonging the survival of rat granulocytes.

Deregulation of the CEACAM Expression Pattern Causes Undifferentiated Cell Growth in Human Lung Adenocarcinoma Cells

CEACAM1, CEA/CEACAM5, and CEACAM6 are cell adhesion molecules (CAMs) of the carcinoembryonic antigen (CEA) family that have been shown to be deregulated in lung cancer and in up to 50% of all human cancers. However, little is known about the functional impact of these molecules on undifferentiated cell growth and tumor progression. Here we demonstrate that cell surface expression of CEACAM1 on confluent A549 human lung adenocarcinoma cells plays a critical role in differentiated, contact-inhibited cell growth. Interestingly, CEACAM1-L, but not CEACAM1-S, negatively regulates proliferation via its ITIM domain, while in proliferating cells no CEACAM expression is detectable. Furthermore, we show for the first time that CEACAM6 acts as an inducer of cellular proliferation in A549 cells, likely by interfering with the contact-inhibiting signal triggered by CEACAM1-4L, leading to undifferentiated anchorage-independent cell growth. We also found that A549 cells expressed significant amounts of non-membrane anchored variants of CEACAM5 and CEACAM6, representing a putative source for the increased CEACAM5/6 serum levels frequently found in lung cancer patients. Taken together, our data suggest that post-confluent contact inhibition is established and maintained by CEACAM1-4L, but disturbances of CEACAM1 signalling by CEACAM1-4S and other CEACAMs lead to undifferentiated cell growth and malignant transformation.

The tyrosine kinase NPM-ALK, associated with anaplastic large cell lymphoma, binds the intracellular domain of the surface receptor CD30 but is not activated by CD30 stimulation

The heterogenous group of anaplastic large cell lymphomas (ALCLs) is characterized by expression of the Ki-1/CD30 antigen, a member of the tumor necrosis factor receptor superfamily. About 40 to 50% of cases diagnosed as ALCL contain a specific chromosomal rearrangement, t(2;5)(p23;q35), resulting in expression of the chimeric tyrosine kinase NPM-ALK. As NPM-ALK-positive lymphomas define a distinct subtype within the group of ALCL, the chimeric protein might be responsible for certain pathogenetic and clinicopathologic characteristics. To better elucidate the function of NPM-ALK, we investigated a possible mechanism for regulation of its activity. We demonstrate that NPM-ALK specifically binds to the intracellular domain of the cytokine receptor CD30. In vitro binding assays revealed that the ALK portion of NPM-ALK mediates interaction of the two proteins. Stimulation of the CD30 receptor by cross-linking with immobilized anti-CD30 antibody results in complete growth inhibition of Karpas 299, an NPM-ALK-positive ALCL cell line, but does not alter proliferation of HDLM-2, a Hodgkins lymphoma-derived cell line lacking t(2;5). Western blot analysis of coimmunoprecipitated CD30 and NPM-ALK proteins from stimulated Karpas 299 cells showed that the interaction of the proteins is not modified by stimulation. Activation of CD30 neither enhanced NPM-ALK activity measured by autophosphorylation of the chimeric tyrosine kinase nor phosphorylation of phospholipase C-gamma, an NPM-ALK substrate. We conclude that NPM-ALK is not stimulated by CD30 activation, but exists as a constitutively hyperactivated protein. Interaction with CD30 may extend the subcellular localization of NPM-ALK to the microenvironment of membrane-associated proteins.

Computational Analysis of Isoform-Specific Signal Regulation by CEACAM1—A Cell Adhesion Molecule Expressed in PC12 Cells

Abstract: CEACAM1 is a signal‐regulating, homophilic cell adhesion receptor system expressed in epithelia, vessel endothelia, and leukocytes. Here, we demonstrate that CEACAM1 is expressed also in PC12 cells, both as the common transmembrane isoforms, CEACAM1‐L and CEACAM1‐S, and as a novel, secreted, differentially spliced isoform. CEACAM1 can have both positive and negative effects on cell signaling. In an attempt to explain this dual behavior, we have initiated computational analysis of the signal‐regulating effects of CEACAM1. This suggests that CEACAM1 can exert its signal‐regulating activities by discriminating between binding of Src kinases and SHP phosphatases, respectively. Major factors that regulate this discrimination are the expression levels and expression ratios of transmembrane CEACAM1‐L and CEACAM1‐S, the concentration of secreted CEACAM1, and homophilic binding of CEACAM1 presented by neighboring cells.

Carcinoembryonic antigen (CEA)-related cell adhesion molecules are co-expressed in the human lung and their expression can be modulated in bronchial epithelial cells by non-typable Haemophilus influenzae, Moraxella catarrhalis, TLR3, and type I and II interferons

BackgroundThe carcinoembryonic antigen (CEA)-related cell adhesion molecules CEACAM1 (BGP, CD66a), CEACAM5 (CEA, CD66e) and CEACAM6 (NCA, CD66c) are expressed in human lung. They play a role in innate and adaptive immunity and are targets for various bacterial and viral adhesins. Two pathogens that colonize the normally sterile lower respiratory tract in patients with chronic obstructive pulmonary disease (COPD) are non-typable Haemophilus influenzae (NTHI) and Moraxella catarrhalis. Both pathogens bind to CEACAMs and elicit a variety of cellular reactions, including bacterial internalization, cell adhesion and apoptosis.MethodsTo analyze the (co-) expression of CEACAM1, CEACAM5 and CEACAM6 in different lung tissues with respect to COPD, smoking status and granulocyte infiltration, immunohistochemically stained paraffin sections of 19 donors were studied. To address short-term effects of cigarette smoke and acute inflammation, transcriptional regulation of CEACAM5, CEACAM6 and different CEACAM1 isoforms by cigarette smoke extract, interferons, Toll-like receptor agonists, and bacteria was tested in normal human bronchial epithelial (NHBE) cells by quantitative PCR. Corresponding CEACAM protein levels were determined by flow cytometry.ResultsImmunohistochemical analysis of lung sections showed the most frequent and intense staining for CEACAM1, CEACAM5 and CEACAM6 in bronchial and alveolar epithelium, but revealed no significant differences in connection with COPD, smoking status and granulocyte infiltration. In NHBE cells, mRNA expression of CEACAM1 isoforms CEACAM1-4L, CEACAM1-4S, CEACAM1-3L and CEACAM1-3S were up-regulated by interferons alpha, beta and gamma, as well as the TLR3 agonist polyinosinic:polycytidylic acid (poly I:C). Interferon-gamma also increased CEACAM5 expression. These results were confirmed on protein level by FACS analysis. Importantly, also NTHI and M. catarrhalis increased CEACAM1 mRNA levels. This effect was independent of the ability to bind to CEACAM1. The expression of CEACAM6 was not affected by any treatment or bacterial infection.ConclusionsWhile we did not find a direct correlation between CEACAM1 expression and COPD, the COPD-associated bacteria NTHi and M. catarrhalis were able to increase the expression of their own receptor on host cells. Further, the data suggest a role for CEACAM1 and CEACAM5 in the phenomenon of increased host susceptibility to bacterial infection upon viral challenge in the human respiratory tract.

CEACAM1-3S Drives Melanoma Cells into NK Cell-Mediated Cytolysis and Enhances Patient Survival.

CEACAM1 is a widely expressed multifunctional cell-cell adhesion protein reported to serve as a poor prognosis marker in melanoma patients. In this study, we examine the functional and clinical contributions of the four splice isoforms of CEACAM1. Specifically, we present in vitro and in vivo evidence that they affect melanoma progression and immune surveillance in a negative or positive manner that is isoform specific in action. In contrast with isoforms CEACAM1-4S and CEACAM1-4L, expression of isoforms CEACAM1-3S and CEACAM1-3L is induced during disease progression shown to correlate with clinical stage. Unexpectedly, overall survival was prolonged in patients with advanced melanomas expressing CEACAM1-3S. The favorable effects of CEACAM1-3S related to enhanced immunogenicity, which was mediated by cell surface upregulation of NKG2D receptor ligands, thereby sensitizing melanoma cells to lysis by natural killer cells. Conversely, CEACAM1-4L downregulated cell surface levels of the NKG2D ligands MICA and ULBP2 by enhanced shedding, thereby promoting malignant character. Overall, our results define the splice isoform-specific immunomodulatory and cell biologic functions of CEACAM1 in melanoma pathogenesis.