The human pathogens N. gonorrhoeae, N. meningitidis, M. catarrhalis, and H. influenzae avoid acquired immune defenses of their host by sophisticated mechanisms such as variation of surface structures or secretion of IgA-specific proteases. However, despite widespread colonization with some of these microorganisms, severe disease outcomes occur only in a small portion of the host population, suggesting that innate immune defenses are able to contain the bacteria in most cases. We demonstrate here that human granulocytes not only recognize CEACAM-binding variants of these pathogens but that bacterial engagement of a granulocyte-specific member of the CEACAM family, CEACAM3, initiates phagocyte effector mechanisms leading to the elimination of the microbes.
It has been observed previously that human granulocytes can recognize opaque variants of pathogenic Neisseriae
in an opsonin-independent manner (29
) and that this recognition depends on members of the CEACAM family (4
). Overexpression of individual CEACAM family members in nonphagocytic cells has revealed that several CEACAMs can mediate uptake in such a cellular background (11
). However, the role of individual receptors in the interaction of granulocytes with CEACAM-binding pathogens has not been analyzed. The data presented in this study provide for the first time biochemical and functional evidence that CEACAM3 is the predominant CEACAM family member on human granulocytes mediating elimination of CEACAM-binding bacteria. Since there is no endogenous ligand known for CEACAM3 (13
), the recognition of pathogenic Haemophilus
, and Neisseria
species now assigns a function to this orphan receptor of the innate immune system.
The efficient CEACAM3-mediated clearance of bacterial pathogens seems to be closely linked to the ability of this receptor to stimulate the small GTPase Rac, a critical regulator of actin polymerization, phagocytosis, and the oxidative response of granulocytes (30
). Though CEACAM6 can mediate internalization when overexpressed in epithelial cell lines (11
), bacterial engagement of CEACAM6 did not lead to Rac stimulation, and interference with CEACAM6 did not block the clearance of CEACAM-binding bacteria by human granulocytes. The difference between CEACAM3- and CEACAM6-mediated events is due to the presence of an ITAM-like sequence in the CEACAM3 cytoplasmic tail. ITAMs have been characterized in several receptors belonging to the Ig superfamily (31
). ITAM function depends on the phosphorylation of key tyrosine residues that is often mediated by Src family protein tyrosine kinases (32
). Interestingly, tyrosine residue Y241 within the CEACAM3 ITAM-like sequence is embedded in the context of YxxM, where a methionine residue is located at the +3 position with respect to the tyrosine (compared with YxxL/I in the canonical ITAM [31
]). Such a YxxM sequence has been shown to serve as a preferred docking site for SH2 domains of the p85 subunit of phosphatidylinositol 3′-kinase (PI-3K) (33
). Therefore, we would predict that phosphorylated Y241 connects phosphorylation of CEACAM3 to the reported recruitment of PI-3K (34
). Since the products of PI-3K can activate several Rac GEFs, such as Vav and SWAP70 (35
), PI-3K binding is a potential molecular link between CEACAM3 ITAM phosphorylation and downstream Rac stimulation.
CEACAM3 does not engage in homo- or heterotypic interactions with other CEACAM family members, and endogenous ligands are unknown (37
). In light of the results presented in this paper, CEACAM3 is best viewed as a specifically adapted single chain phagocytic receptor involved in the clearance of CEACAM-binding bacteria by human granulocytes. Most of the known CEACAM-binding bacterial species are human-specific pathogens. Likewise, the expression of CEACAM3 is also restricted to human granulocytes, and no homologues of CEACAM3 have been detected in rodents or nonhuman primates (38
), suggesting a striking coevolution of human innate immune defenses with a restricted set of specialized pathogens. It is tempting to speculate that gonorrhea-associated complications effecting reproduction such as early trimester abortion or infertility (40
) might have provided a selective advantage to individuals able to counteract CEACAM-binding bacteria by innate immune defenses. On the other hand, pathogens might evolve variants of their CEACAM-binding adhesins that retain association with CEACAM family members expressed on epithelial cells (such as CEA or CEACAM1) but that loose recognition by CEACAM3. Such adhesins would allow attachment to and invasion of epithelial cells but would avoid elimination by granulocytes. Indeed, several meningococcal and gonococcal OpaCEA
proteins have been characterized that exhibit this property (11
). It will be interesting to investigate whether the ability to cause severe forms of disease is correlated with the expression of CEACAM-binding adhesins that are not recognized by CEACAM3.
Together, the results presented in this paper describe the orphan receptor CEACAM3 as a novel single-chain phagocytic receptor of the innate immune system. Since internalization via CEACAM3 is opsonin independent, this receptor should be of particular importance in the control of bacteria that do not induce protective acquired immune responses. Since multiple commensal and pathogenic bacteria found in association with human mucosal surfaces are exploiting CEACAM family members (43
), CEACAM3-mediated, opsonin-independent phagocytosis by granulocytes might be a general defense mechanism evolved by the human innate immune system directed against CEACAM-binding microorganisms.