Expression of C-Reactive Protein in the Human Respiratory Tract

C-reactive protein (CRP) is a constituent of normal human serum (NHS) (33). The concentration of CRP in serum is generally less than 2 μg/ml but increases by as much as 1,000-fold in response to a stimulus such as tissue injury or inflammation (5). Following removal of the inflammatory stimulus, CRP levels decline rapidly. These features have made CRP useful as a clinical marker of an inflammatory process, although the function of this acute-phase reactant and its precise role in host defense remain poorly understood. Until recently there had been no demonstration of a direct antimicrobial effect of CRP in vitro, and the study of CRP using animal models has been limited by the marked differences in the regulation of CRP expression in animals compared to humans (27, 46). There is, however, evidence to suggest that CRP contributes to innate immunity. Mice, which have a constitutively low level of CRP expression, are more resistant to experimental pneumococcal sepsis when carrying the human CRP transgene conferring inducible high-level expression as in humans (34). The protective effect of CRP is thought to be mediated by its ability to act as an opsonin and, when bound, to activate the complement by the classical pathway through interaction with complement component C1q (16, 37). The CRP transgene reduces bacteremia following an intraperitoneal inoculation of pneumococci in both decomplemented and complement-expressing mice, suggesting that there is also a complement-independent pathway for CRP-mediated protection, perhaps through direct opsonization (35). CRP received its name because it binds to the C polysaccharide or cell wall teichoic acid of Streptococcus pneumoniae. It is now known that CRP binds in a calcium-dependent manner to choline phosphate or phosphorylcholine (ChoP) residues found on C polysaccharide (38). ChoP had been considered to be a highly unusual structural feature in prokaryotes. It is now clear that in addition to S. pneumoniae, many of the bacterial species that normally inhabit the respiratory tract express ChoP, the molecular target of CRP, on their cell surface. These species are now known to include Streptococcus oralis and S. mitis, Haemophilus influenzae and H. somnus, Actinobacillus actinomycetemcomitans, Fusobacterium nucleatum, various Actinomyces species, the commensal Neisseria species, and Mycoplasma species such as M. fermentans (8, 14, 24, 29, 30, 41, 45). The presence of ChoP on a large and diverse collection of species found primarily on the mucosal surface of the airway including gram-positive and gram-negative bacteria, as well as Mollicutes, and its absence from species residing outside the respiratory tract, suggests that this structure contributes to survival in this host environment. Data from both animal models of nasopharyngeal carriage and natural lower respiratory tract infection of humans suggest that ChoP, while not a requirement for survival within the respiratory tract, contributes to the ability of bacteria to persist at these sites (44, 45). The expression of ChoP has been shown to confer resistance to antimicrobial peptides found on the mucosal surface of the upper respiratory tract, such as LL-37, that target structural differences in membranes between host and microbial cells (21). In this regard, the expression of ChoP may allow bacteria to mimic the same structure found on all eukaryotic membrane lipids in the form of phosphatidylcholine. ChoP also allows for bacterial invasion of epithelial cells through interaction with the receptor for platelet-activating factor (rPAF), whose natural ligand, PAF, also contains ChoP (8, 32). CRP, therefore, may contribute to innate immunity by specifically targeting this virulence determinant common to many of the bacterial pathogens of the respiratory tract. A direct antimicrobial effect of CRP, however, has been demonstrated only in the case of H. influenzae, where concentrations of the protein as low as 10 ng/ml bind to ChoP and mediate a complement-dependent bactericidal effect (44). Another striking feature of the ChoP ligand is that there is phase variation in the amount or presence on the bacterial cell surface in many of the species expressing this moiety (17, 40, 42, 45). For H. influenzae, only those phase variants expressing ChoP on their lipopolysaccharide are sensitive to the bactericidal effects of human serum CRP (44). This suggests that these pathogens have developed an efficient means for evasion of clearance mechanisms such as that involving CRP that specifically targets ChoP. The interplay between the expression of ChoP and local amounts of CRP, therefore, may function to maintain the commensal state and limit the pathogenicity of many important respiratory tract bacteria. Human serum CRP is a cyclic pentameric protein of five identical nonglycosylated subunits of 206 amino acids, each with a molecular mass of 24 kDa, that are noncovalently bound to form the mature CRP molecule (13). Serum CRP is synthesized by hepatocytes in the liver as a single-chain precursor with a cleavable signal sequence at the N terminus (36). CRP was initially thought to be produced and secreted only by hepatocytes under induction primarily by interleukin-6 (IL-6), with a synergistic effect of IL- 1 (39, 48). Tumor necrosis factor alpha, transforming growth factor β, and IL-11 have also been shown to affect hepatic CRP expression (3, 4, 10, 15, 23). There is also evidence of CRP expression by Kupffer cells and peripheral blood mononuclear cells, where it was shown to be a membrane protein that is not secreted (12, 19). Although the source and regulation of serum CRP have been extensively studied, the expression of CRP in the human respiratory tract, particularly the heavily colonized upper respiratory tract where organisms bearing its ChoP target reside, has not been addressed. In this report, we show that CRP is found in secretions from the human airway and that epithelial cells lining the mucosal surface of the upper respiratory tract may be a source of this protein. Furthermore, we demonstrate that CRP isolated from the mucosal surface of the airway has antimicrobial activity.