Hematopoietic CEACAMs (Granulocytes)

While most primordial CEACAMs are found on epithelia, of this group only CEACAM1, which gave rise to the current expansion of this family in mammals, is found on hematopoietic cells as well. CEACAM1 is again the most abundant CEACAM-family member in the hematopoietic system, being expressed on T-cells, B-cells, platelets and granulocytes. Its almost ubiquitous expression made CEACAM1 the CEACAM protein most extensively researched. Due to the ITIM in the cytoplasmatic domain of the L-spliceforms, CEACAM1 was studied in regard of its immunoreceptor-like signalling capabilities, and indeed, CEACAM1 is able to mediate inhibitory signalling in a number of scenarios. In the hematopoietic system, CEACAM1 not only serves as a receptor for pathogens exploiting epithelial CEACAMs for colonisation of their host, but modulates immune responses in several settings (Gray-Owen, Blumberg, 2006).

In resting granulocytes, CEACAM1 is able to delay spontaneous apoptosis of these short lived leukocytes (Singer et al., 2005) via an SHP-1 and Erk1/2 mediated pathway. Interestingly, in a B-cell model CEACAM1 was found to promote apoptosis when challenged with opaque gonococci (Pantelic et al., 2005) leading to inhibition of antibody production. This effect does not involve SHP-1, Syk or PLC but relies in part on activity of SHIP or BTK. In a human B-cell line, Lobo et al demonstrated that CEACAM1 can serve as a negative co-receptor for the BCR and regulates phosphatidylinositol 3-kinase (PI3K) activity (Lobo et al., 2009).

CEACAM1 can inhibit several ITAM-based signals via its ITIM-sequence and seems to play an important role for mucosal pathogens to suppress immune responses.

This includes TLR2 mediated signalling (Slevogt et al., 2008), which is suppressed by UspA1 expressing Moraxella catarrhalis or Opa expressing N. meningitidis. This suppressive effect may be of benefit to several CEACAM-binding urogenital and mucosal pathogens in the first step of infection, since CEACAM1 is also found on those epithelia.

The inhibitory effects of CEACAM1-L on T-cells have been subject to several studies. N. gonorrhoeae is able to suppress activation of CD4⁺ T-cells by recruiting

Carcinoembryonic-antigen related Cell Adhesion Molecules (CEACAMs)

CEACAM1 to the cell surface and without getting phagocytosed (Lee et al., 2008) and N. meningitidis or membrane vesicles released by that pathogen seem to be able to confer this effect as well (Lee et al., 2007). The view, that CEACAM1-binding must lead to inhibition of T-cell proliferation has been challenged though, since another study showed that CEACAM1-binding as well as non-binding bacteria or membrane vesicles exhibit a positive effect on proliferation of T-cells (Youssef et al., 2009). Differences in experimental setups may account for some of the discrepancies, but still CEACAM1 seems to be the most complex CEACAM in terms of variability and signalling properties that depend not only on the pre-activation status of the cell, but also on the CEACAM1 isoforms expressed and their expression ratios (Gray-Owen, Blumberg, 2006). A humanised mouse model, which expresses human CEACAM1 in addition to the endogenous mouse CEACAM1, has been established lately, allowing the study of pathogenic interactions with immune-cells expressing this CEACAM-family member only (Gu et al., 2010). This model may shed some more light on the contributions of single CEACAMs in the response to human-specific pathogens as N. meningitidis or N. gonorrhoeae. Another disease model that is available in mice is mimicking multiple sclerosis. In this model, blockage of CEACAM1 by antibodies increased severity of the disease while cross-linking CEACAM1 by addition of a CEACAM1-Fc chimera ameliorated the course of the myelitis by inhibiting IFNγ and IL17 production. This inhibition was found to be mediated by invariant natural killer cells, as mice that do not have these cells were unresponsive to the treatment (Fujita et al., 2009).

CEACAM8 is a granulocyte-specific member of the CEACAM-family. It is linked to the membrane via a GPI-anchor and released to the cell surface upon activation of the granulocyte. Although related to CEACAM1 evolutionarily, CEACAM8 does not recognize pathogens like N. gonorrhoeae, but engages in heterophilic interactions with CEACAM6. Another known ligand of CEACAM8 is galectin-3 (Yoon et al., 2007), a protein associated with IgE binding, which might point to a role of CEACAM8 in response to parasites. Engagement of CEACAM8 leads to de-granulation and strong adhesion of granulocytes.

CEACAM6, which can also be found on epithelial tissues, is expressed on granulocytes as well. On these cells, it may mediate phagocytosis of OpaCEA

expressing gonococci (Gray-Owen et al., 1997a) and its stimulation contributes to

Introduction

Carcinoembryonic-antigen related Cell Adhesion Molecules (CEACAMs)

Skubitz, Skubitz, 2010). As on epithelia, CEACAM6 in granulocytes is a receptor for Afa/Dr adhesins of diffusely adhering E. coli (DAEC) (Berger et al., 2004) and has been linked to inflammatory processes induced by type1 pili of E. coli, probably contributing to inflammatory bowel disease like Crohn's disease (Semiramoth et al., 2008).

CEACAM4 is likely to be the first ITAM-carrying offspring of CEACAM1. Although capable of inducing phagocytosis via its cytoplasmic domain (unpublished results), no ligand for its single extracellular IgV-like domain has been identified so far (Popp et al., 1999). Since its initial description in 1991 (Kuroki et al., 1991) it has been subject to very few studies, most of the time as a comparison to its fellow family members.

CEACAM3 is an exceptional member of the CEACAM-family, not by its signalling variability or complexity like CEACAM1, but by its simplicity and effectiveness with which it fulfils its task. CEACAM3 expression is limited to granulocytes and it is the less abundant CEACAM on these cells. It does not engage in homo- or heterophilic interactions, but seems to represent one side of an paired receptor system with CEACAM1.

CEACAM3 is a decoy and represents a means of the innate immune system to fight off CEACAM-binding pathogens by mediating efficient uptake and killing (Chen, Gotschlich, 1996). But not all CEACAM-binding pathogens are recognised by CEACAM3. N. meningitidis, for example, only interacts with CEACAM1.

N. gonorrhoeae though expresses several colony opacity associated (Opa) proteins that are recognised by CEACAM3 as well. In a chicken B-cell model (DT-40), engagement of CEACAM3 by gonococci will lead to cell death of the infected cells (Chen et al., 2001a). If this finding mirrors the processes in human granulocytes needs to be questioned, though. Not only because in human granulocytes CEACAM1 might interfere with apoptosis as mentioned before, but also based on the finding, that Syk is required for uptake by B-cells, which was shown to be

Figure 3.1.4: CEACAMs can mediate phagocytosis of pathogens that usually exploit CEACAMs on epithelia for colonisation of their host, but pathogens may turn CEACAM1 binding into another immune evasive strategy by suppressing the T-cell response (Hauck and Mayer 2003)

Carcinoembryonic-antigen related Cell Adhesion Molecules (CEACAMs)

CEACAM3 does not display a broad spectrum of signalling functions as observed for CEACAM1, which relates to its limited expression pattern and narrow ligand specificity. Since this property will keep crosstalk of signalling events low, circumstances for detailed analysis of the underlying signal-transduction cascade are very favourable.

While in mice, CEACAM1s N-terminal domain was altered due to evolutionary pressure in order to disallow binding of a viral pathogen, in humans, the bacterial pathogen-binding domain was linked to an intracellular signalling apparatus that will facilitate not only uptake, but also killing of the pathogen. In dogs a similar couple arose during the radiation of CEACAM1-related genes, giving rise to CEACAM28, an ITAM-containing receptor expressed on cells of the immune-system. The recent discovery of this CEACAM-family member raises the question for possible dog-specific pathogens or immune-regulatory affairs that are addressed by this receptor.

Introduction

CEACAMs: their signalling properties and related pathways