PERSPECTIVE
Modulation of Host-Bacterial Interactions by Collectins

Erika C. Crouch

Department of Pathology, Washington University School of Medicine, St. Louis, Missouri

Pulmonary defenses against inhaled bacteria are complex, overlapping, and mechanistically heterogeneous. These include physical barriers and a diversity of biochemical and cellular defenses. Acquired humoral and cellular immunity are of obvious importance. However, innate, nonclonal mechanisms play important roles, particularly in the interval before the development of specific cellular immunity and in various immunodeficiency states. In the absence of specific immunity, innate mechanisms often determine whether bacterial inhalation is followed by immediate clearance, colonization with asymptomatic carriage, mucosal infection, tissue invasion, or dissemination and sepsis. In normal hosts and in the absence of exposure to unusually large numbers of virulent organisms, these natural front-line defenses appear sufficient to allow rapid bacterial clearance without the development of infection.

Innate pulmonary immunity includes cell-mediated mechanisms that involve the activities of the resident and recruited phagocytes. In addition, there are a wide variety of constitutively or readily inducible proteins and peptides with antibacterial activities in vitro that accumulate in the lung in vivo. These substances are synthesized by epithelial cells, which line the airways and alveoli, and resident leukocytes. Putative pulmonary host-defense molecules include:

 cell wall hydrolases; notably, lysozyme

 iron-binding proteins, such as lactoferrin and trans- ferrin

 complement components

 defensins and cathelicidins

 "matrix molecules," such as cellular fibronectin

 antiproteases, such as antileukoproteinase

 proteases, such as matrilysin, and

 surfactant proteins A and D (SP-A and SP-D).

Lung Collectins

SP-A and SP-D are structurally homologous members of a family of collagenous, calcium-dependent lectins, which includes serum mannose-binding lectin (MBL) (1). Human lung collectins are synthesized by alveolar epithelial cells, nonciliated bronchiolar cells, some epithelial cells lining the larger airways, and glands associated with the upper respiratory and aerodigestive tracts (1, 2). Both collectins show specific saccharide-dependent interactions with a variety of bacterial, fungal, and viral organisms, and show specific binding to leukocytes and/or lung epithelial cells in vitro (1, 3).

Host Defense Roles of Collectins

In many cases, interactions of collectins with microorganisms involve specific binding of the lectin domains of the collectin to glycoconjugates integral to the microbial cell wall or envelope. Although the collectins often interact with the same microbial surface structures, their specific sites of binding may differ (e.g., SP-A binds to the lipid A domain of a lipopolysaccharide [LPS], whereas SP-D binds to core saccharide residues) (4). The collectins may also interact with different surface structures on the same organism (e.g., SP-A binds to capsular polysaccharides on specific strains of Klebsiella pneumoniae, whereas SP-D binds to LPS) (7). In addition, some interactions are mediated by the binding of microbial lectins to the collectin. For example, influenza A and Herpes simplex viruses bind specifically to N-linked sugars on SP-A (8). Thus, there is a potential for cooperative, competing, or collectin-specific interactions in vivo.

On the basis of these observations, a wide variety of potential "antibacterial" defense functions have been suggested for lung collectins, including opsonization, microbial agglutination with effects on cellular uptake or mucociliary clearance, microbial recognition, modulation of leukocyte activation, detoxification or presentation of endotoxin, regulation of cytokine production, phagocyte recruitment, and interference with bacterial growth or epithelial adherence (1, 2, 9, 10). A number of potentially deleterious activities have also been suggested: competition with phagocyte lectins, inhibition or inappropriate activation of LPS-activated defenses, excessive amplification of inflammatory reactions, enhanced internalization of organisms that are ineffectively killed, and air-space retention of a microorganism (1, 11).

At present, the most compelling evidence for pulmonary collectin-mediated, antibacterial host-defense activity comes from characterization of SP-A-null, transgenic mice (12). Although the animals showed no discernible abnormality in surfactant function or homeostasis (15), host-defense defects have been observed after microbial challenge. For example, SP-A-null mice show increased proliferation and dissemination and decreased phagocytosis of Group B streptococci (GBS) strains, and decreased clearance of Staphylococcus aureas and Pseudomonas aeruginosa after intratracheal inoculation. In all these studies, SP-A-null animals inoculated in the presence of purified SP-A demonstrated a wild-type phenotype. It has not, however, been established that SP-A is functioning as an opsonin. Other mechanisms, such as modulation of macrophage mannose receptor expression, could be involved (16).

SP-D Interactions with Gram-Negative Bacteria

SP-D is known to interact with the LPS of gram-negative bacteria, can mediate bacterial agglutination, and has been shown to enhance the phagocytosis of gram-negative and gram-positive bacteria by human neutrophils in vitro (4, 17). However, there are no published data that convincingly demonstrate the opsonization (i.e., increased internalization of bacteria with surface-associated SP-D) and enhanced killing of gram-negative organisms by SP-D. In fact, Pikaar and coworkers reported that SP-D does not enhance the internalization of a laboratory strain of E. coli shown to be aggregated by SP-D (18). Given these activities and the close structural homology with other collectins, we have hypothesized that SP-D contributes to antibacterial host defense or inflammatory and immune regulation in the lung.

SP-D Interactions with Pseudomonas aeruginosa

In this issue, Restrepo and coworkers describe the interactions of rat SP-D with two strains of P. aeruginosa, one mucoid and one nonmucoid (19). They observed lectin-dependent binding of SP-D to these organisms, as well as an approximately twofold, concentration-dependent enhancement of internalization. Under these conditions, little enhancement of internalization of a strain of Haemophilus influenzae was observed. Opsonization experiments, in which the organisms were preincubated with SP-D and washed before their addition to the cells, resulted in comparable enhancement in phagocytosis. However, there was no increase in the uptake of nonopsonized or opsonized organisms when the macrophages were attached to SP-D- coated slides. Internalization of the organism was accompanied by a significant decrease in colony-forming units for the mucoid, but not the nonmucoid strain, suggesting that the nonmucoid organisms were internalized but not killed. Interestingly, SP-D did not aggregate either strain, despite efficient aggregation of control E. coli. Thus, the studies provide evidence for SP-D lectin-dependent, receptor-mediated opsonization and killing of a mucoid strain of P. aeruginosa.

Potential Clinical Implications

The previously mentioned studies are of particular interest given the recognized importance of mucoid strains of P. aeruginosa for the pathogenesis of chronic lung disease associated with cystic fibrosis. Although recent studies have focused on the possible role of altered ionic conditions on the activity of defensins and other host-defense proteins, the possible contributions of deficient collectin activity merits careful investigation. In this regard, the levels of SP-A and SP-D have been reported to be decreased in the lung lavages of at least some patients with cystic fibrosis (20). Given the importance of P. aeruginosa in hospital-acquired pneumonia and sepsis, the studies could have much broader implications.

SP-D Receptors

The existence of one or more SP-D receptors on leukocytes was first suggested by studies demonstrating a potent chemotactic activity of SP-D for neutrophils and monocytic cells (21, 22), and more recently, by SP-D-dependent alterations in actin polymerization in alveolar macrophages (23). However, the study by Restrepo and associates is the first to implicate a SP-D receptor in bacterial phagocytosis.

The specific mechanism of cellular binding remains to be elucidated. Calcium-dependent and calcium-independent interactions with monocyte-macrophages have been described (24). The lectin domain of SP-D can interact in a calcium-dependent fashion with glycoconjugates expressed on the macrophage surface, and there is evidence that SP-D- stimulated monocyte chemotaxis involves such a mechanism (21, 25).

The only characterized SP-D-binding protein is gp-340, a member of the scavenger receptor superfamily, which binds to the carboxy-terminal domain of SP-D via a calcium- dependent mechanism (26). Although gp-340 has been immunologically localized at the surface of alveolar macrophages, it has not yet been shown to mediate SP-D binding to these cells. Two C1q-binding proteins (C1qR and C1qRp) known to bind to SP-A and other collectins do not bind to SP-D (3). Specific interactions with other SP-A receptors, including a 210-kD receptor implicated in SP-A-mediated phagocytosis of Mycobacterium bovis (27), have not yet been described.

Environmental Modulation of the Bacterial Phenotype

There are numerous microbial determinants of host-bacterial interactions. Determinants include not only the innoculum and route of delivery, but also the action of various virulence factors (28). Among these are adhesins that mediate epithelial attachment. These include attachment proteins associated with pili or flagella and integral cell-wall components, such as specific LPS or capsular polysaccharides. Bacterial pathogens may also elaborate factors required for epithelial invasion (invasins), proteases or toxins that facilitate tissue invasion and spreading through tissue destruction (aggressins), and factors that inhibit host defense without significant associated tissue damage (impedins) (29).

It is increasingly appreciated that environmental conditions, in vitro or in vivo, can modify the bacterial phenotype and expression of these molecules, thereby altering the organism's potential for adhesion, colonization, or invasion. Bacterial phase variation, the spontaneous "on-off" of specific bacterial genes, allows the rapid selection of variant organisms expressing virulence factors suitable for specific biologic niches (30). In many cases, the development of lung infection in vivo involves a step-wise process whereby organisms derived from the skin or GI tract first colonize biofilms associated with upper-airway compartments. Further adaptations and selection of alternative phase variants may be required for colonization of the much different and "effectively" sterile environment of the distal airways and alveoli. Still, additional selection events may contribute to mucosal invasion and the evasion of host defenses. Thus, the use of only two, nonisogenic, mucoid, and nonmucoid strains is a potential limitation of this study.

Observed differences in SP-D binding and killing of mucoid and nonmucoid strains and their corresponding heat-killed organisms are not readily explained. Are differences attributable to the presence or absence of a capsule, or to other differences in bacterial structure? The only established ligand for SP-D on gram-negative organisms is LPS (1), and binding to Pseudomonas LPS has been demonstrated (31). Because abundant mucoid exopolysaccharides might mask LPS-binding sites, the efficient binding of SP-D to the mucoid strain might suggest interactions with the mucinous exopolysaccharides. On the other hand, some isolates of P. aeruginosa from cystic fibrosis patients have been reported to be deficient in O-polysaccharides (32), which might favor binding of SP-D to its binding sites to the core oligosaccharide. In any case, these studies further emphasize the potential importance of variably expressed surface structures in determining the interactions of bacteria with host-defense molecules and cells.

Environmental Modulation of Lung Lectin Function

Environmental factors could also influence the function of SP-D and other lung lectins. For example, Reading and coworkers have shown that the infectivity of SP-D-sensitive strains of influenza A virus is increased in hyperglycemic, diabetic mice in vivo (33). These effects were observed at glucose concentrations that inhibited the ability of SP-D to inhibit infectivity in vitro. Thus, inhibition of pulmonary lectins could account for the increased risk of diabetic patients for certain bacterial and viral respiratory infections. Changes in the microenvironmental milieu at sites of bacterial colonization and infection could similarly modulate function by modifying the concentrations of various endogenous ligands, such as phosphatidylinositol or the local accumulation of soluble, bacterial glycoconjugates. Recently, Mariencheck and coworkers described the degradation of SP-A by a P. aeruginosa-derived protease (34).

Molecular Complexity of the Lung's Host Defense System

The list of potential host-defense molecules is obviously large and almost certainly incomplete. Many of the molecules listed previously are elaborated at multiple levels of the respiratory tract, in a variety of biochemical or cellular milieus, and probably in differing amounts at different anatomic sites. Some molecules are constitutively secreted, whereas others are constitutively synthesized but subject to regulated secretion. In some cases, proteins are constitutively synthesized and secreted at some sites, yet subject to regulated secretion from glandular or surface epithelial cells. Many factors are induced or significantly amplified in the setting of lung injury and/or after exposure to microbial products. There is also considerable overlap in the types of host-defense molecules synthesized by parenchymal cells and resident or recruited leukocytes.

It could prove problematic to discern the relative importance of various defense proteins for different microorganisms, even using contemporary transgenic technologies. Intratracheal challenge models in transgenic animals deficient in putative host-defense proteins can identify defects and their mechanisms, but cannot readily establish the biologic importance of the defect. Massive inoculation of cultured bacteria to the conducting airways will likely bypass the processes of phase variation relevant to the more usual sequence of colonization and infection. Models of spontaneous infection will almost certainly be needed.

Because many of the known host-defense proteins and peptides demonstrate antibacterial activity against the same strains of bacteria in vitro, considerable redundancy seems likely. It is possible that the administration of sufficient concentrations of a number of variety of antibacterial factors might "repair" host-defense defects resulting from a deficiency of a specific host-defense protein.

Models of SP-D Deficiency

Two laboratories have recently described the development of transgenic models of SP-D deficiency (35, 36). Although studies examining the host-defense functions of these animals have not yet been published, the animals show striking perturbations in surfactant homeostasis with accumulations of surfactant lipid, possible alterations in the production of surfactant proteins, abnormalities in the morphology of alveolar macrophages and type II cells, peribronchial infiltrates of lymphocytes, and the development of emphysema. Such changes may alter the host response to inhaled organisms (e.g., through enhanced production or altered activity of other host-defense proteins, antimicrobial effects of surfactant lipids, or prior activation of inflammatory cells) and complicate the interpretation of challenge experiments similar to those used for SP-A.

Future Directions

Important directions for future research include a further elucidation of microbial specificity and cellular consequences of binding and internalization, an examination of potential interactions among antibacterial factors, a more precise delineation of the cellular localization of specific collectins and specific multimeric forms of these molecules, and the characterization of mechanisms of cellular recognition of collectins.

	Footnotes

Address correspondence to: Erika C. Crouch, M.D., Ph.D., Dept. of Pathology, Barnes-Jewish Hospital, 216 S. Kingshighway, St. Louis, MO 63110-1092.

(Received in original form September 19, 1999).

Acknowledgments: This work was supported by the National Institute of Health grants HL29594 and HL44015.

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