What is the real role of antimicrobial polypeptides that can mediate several other inflammatory responses?

Abstract

Antimicrobial peptides are effector molecules of innate immunity with microbicidal and pro- or anti-inflammatory activities. Their role is now widening following evidence that one such multifunctional peptide, LL-37, induces angiogenesis, a process essential for host defense, wound healing, and tissue repair.

The last two decades have heralded impressive progress in the identification of a broad array of structurally and functionally diverse polypeptides implicated in many aspects of the host response to infection and other inflammatory stimuli.

Almost half a century ago, Hirsch described the antimicrobial properties of phagocytin, a crude protein fraction of polymorphonuclear leukocytes (1), thereby setting the stage for a growing interest in the biologic activities of protein components of inflammatory cells. The subsequent exploration of the functional capabilities of defined proteins and peptides in inflammation paralleled the evolving methodology of protein biochemistry and purification, and molecular biology. In this historical context, the initial focus remained on the antibacterial action of newly isolated proteins and peptides, reflecting an ongoing search for new antibiotics and the relative ease of conducting bioassays of bacterial viability (2, 3).

Discovery of proteins and polypeptides with antibacterial properties

Numerous proteins and polypeptides with antimicrobial activity in vitro have now been isolated and/or cloned from a broad range of both simple and complex organisms, including humans. Thus, endogenous-polypeptide antibiotics became prominent targets in the study of antimicrobial host defenses per se and also as potential pharmacologic agents (3). Whole families of such gene products have been identified in plants, insects, and other animals (4) in settings that are consistent with prominent roles in innate immunity. How well have these roles been defined so far?

The initial demonstration of antimicrobial activity of isolated polypeptides in vitro, often under very artificial laboratory conditions and against nonpathogenic strains of bacteria, did not establish whether such polypeptides played a prominent role in vivo. Moreover, since these polypeptides do not act alone and many share the apparent molecular determinants of antimicrobial activity, elucidation of their role in a complex mix in vivo has remained elusive.

Polypeptides with antimicrobial and other biologic activities

Uncertainty grew concerning the actions and effects of individual polypeptides after several were found to possess other biologic activities, apparently unrelated to their antimicrobial actions (Figure 1). For example, α-defensins, at concentrations far lower than are required for their antimicrobial action, are capable of several other functions such as chemotactic and corticostatin-like actions (5).

Figure 1.

Functions of “antimicrobial” polypeptides in innate immunity.

In this issue of the JCI, Koczulla et al. (6) identify yet another novel biologic activity of the polypeptide LL-37/hCAP-18, initially described as an antimicrobial member of the cathelicidin family (7). Cathelicidins, first isolated from polymorphonuclear leukocytes, are also constituents of lining epithelial cells (6). Proteolytic cleavage of cathelicidin proforms is required to free the potent antibacterial activity of the C-terminal peptide LL-37/hCAP-18. The uncleaved proform is devoid of antibacterial activity. However, at sub-nanomolar concentrations, it is capable of blocking the bioactivity of endotoxin (8). Koczulla et al. now show that μg/ml concentrations of synthetic LL-37/hCAP-18 stimulate angiogenesis both in vitro and in vivo (6). The proliferative effect on endothelial cells and vessel formation is convincingly demonstrated under tissue culture conditions. These effects are also manifest in vivo: administration of LL-37 was shown to stimulate capillary formation in a rabbit hind-limb model of ischemia, and mice deficient in the murine analog of LL-37/hCAP-18 demonstrated less neovascularization of skin lesions than wild-type controls. Both the chemoattractant and the angiogenic actions of LL-37 are thought to be receptor-mediated by interaction with formyl peptide receptor–like 1, found on macrophages, neutrophils, and subsets of lymphocytes (6). It is particularly interesting that upon exposure of human umbilical cord endothelial cells to human serum (10%), which was previously shown to inhibit the antimicrobial activity and anti–host cell cytotoxic action of LL-37 (6), the angiogenic activity of LL-37 was unchanged.

The angiogenic activity of exogenous LL-37, as demonstrated in the animal experiments, does not reveal whether endogenous LL-37 participates in the regulation of neovascularization by the many other angiogenic factors that operate in vivo. Also, what governs the release of the active peptide from its precursor during inflammation is largely unknown.

The recent report that the ribonuclease angiogenin, a component of specialized epithelial cells of the small intestinal crypt known as Paneth cells, is also microbicidal (9) further illustrates that such agents may be multifunctional. Obviously, it is a formidable task to define the (patho)physiologic contribution of any individual agent to in vivo events where multiple agonists and antagonists, at different concentrations and with different affinities, compete for the same targets.

Whatever the primary function of these peptides, the study by Koczulla et al. (6) adds to the growing evidence that polypeptides produced, stored, and secreted by inflammatory and epithelial lining cells may have more than one role in the complex innate immune responses of the host. This recognition raises new issues: much of the interest in the pursuit of these polypeptides is linked to the hope that synthetic or recombinant derivatives of bioactive polypeptides, such as LL-37/hCAP-18, may have a future as administered agents for the treatment of infection and its sequelae. However, if such agents participate in more than one biologic activity, possibly at different stages in an inflammatory process, the evaluation of their usefulness and safety becomes accordingly more elaborate.

Is it reasonable, then, to anticipate the addition of such bioactive substances to the pharmacopoeia as treatments of complex clinical disorders? Notwithstanding the unknowns sketched here that require clarification, the answer is probably yes.

Recent reports suggest a role for a cathelicidin common to both mice and humans, in addition to other endogenous polypeptides with antibacterial activity, in providing protection against skin infections (10–12). Other administered proteins and peptides have been shown to protect animals against systemic infections (3, 13).

When disease and/or treatment deplete host stores of proteins and peptides with defense functions and impair or overwhelm their production by both myeloid and epithelial cells, replenishment with exogenous analogs may well become an important adjunctive therapy. This can be envisioned, for example, in cases of otherwise healthy children with severe pediatric bacterial infections that do not respond to available antibiotics (14, 15).