PERSPECTIVE
Recognition of Bacterial Endotoxin in the Lungs

Thomas R. Martin

Pulmonary Research Laboratories, Seattle VA Medical Center, and the Division of Pulmonary and Critical Care Medicine, Department of Medicine, University of Washington School of Medicine, Seattle

The early recognition of bacterial products is critical for our survival. Each day we breathe more than 7,000 liters of air, laden with inorganic and organic particles and an array of microbes. During sleep, we aspirate oropharyngeal secretions containing gram-positive and gram-negative organisms and their products. Patients in the hospital have altered flora in their oropharyngeal and gastric secretions, and some are exposed to therapeutic aerosols carrying environmental microbes. Yet normal humans rarely develop pulmonary infections and most hospitalized patients do not develop nosocomial pneumonia because highly effective host defenses have evolved to protect the lungs from these ubiquitous microbial challenges.

The initial defense of the lungs against the entry of particulates involves the mucociliary system, which lines the nasopharynx and conducting airways, and macrophages that wander the airways and alveolar surface. The mucociliary system removes particulates deposited in the conducting airways, but bacteria and their products that reach the alveolar membrane fall beyond the mucociliary system and are recognized by macrophages, the sentinel cells of the innate immune system in the lungs. Innate immunity provides a means to constantly sample the air-space environment and maintain vigilance for microbial products that reach the alveolar membrane (1).

The innate immune system has both a recognition function, which detects bacterial products in tissues, and an effector function, which attracts phagocytic leukocytes to sites of bacterial entry into tissues. The recognition function is served by soluble proteins that prepare bacterial products for cellular recognition and a family of pattern recognition-receptors on the surface of macrophages and other leukocytes that recognize common bacterial motifs not found on mammalian cells. Pattern-recognition receptors include the mannose receptor, the macrophage scavenger receptor, collectins, integrins, and CD14, which was originally identified as a differentiation marker on monocytes/macrophages (2). The common ligands for pattern-recognition receptors include bacterial cell wall constituents, such as lipopolysaccharides of gram-negative bacteria, lipoteichoic acids of gram-positive bacteria, lipoarabinomannans of mycobacteria, and bacterial-derived DNA and lipoproteins. Macrophage-independent recognition pathways also exist in innate immunity, as short formylated bacterial peptides bind directly to specific receptors on neutrophils that mediate activation and chemotaxis (6, 7).

	Recognition of Bacterial Endotoxin

Bacterial lipopolysaccharide (LPS) (endotoxin) is the prototypical bacterial signal that activates innate immunity, and major advances have occurred in understanding how host cells recognize endotoxin (Figure 1). This new information has major implications for understanding how inflammatory reactions are initiated and perpetuated in the lungs. Bacterial LPS is a component of the gram-negative cell wall, whose structure consists of an acylated diglucosamine head group (lipid A) linked to a chain of repeating disaccharides. The lipid A structure imparts the biologic activity of LPS. The polysaccharide tail imparts the antigenic characteristics of LPS, and varies in LPS from different bacterial species. The acylated lipid A structure initially suggested that LPS might bind directly to biologic membranes and be directly recognized by leukocytes. A consistent and unexplained paradox existed, however, as high concentrations of LPS are required to activate macrophages in vitro (e.g., 1 µg/ml or more in the absence of serum), whereas nanogram concentrations of LPS activate inflammatory responses in vivo and in whole blood ex vivo.

View larger version (21K): [in this window] [in a new window]   Figure 1.   LPS binding to soluble and membrane proteins. LPS in solution binds to LBP, which disaggregates LPS and transfers it to different targets. Transfer of LPS to high density lipoprotein (HDL) inactivates LPS. The LPS/sCD14 complex can activate cells that lack membrane CD14, such as endothelial and epithelial cells. Transfer of LPS to membrane CD14 results in the activation of a second membrane protein, likely TLR4, that initiates intracellular signaling via an IL-1R-like signaling cascade.

This paradox was resolved when Tobias, Ulevitch and coworkers discovered an LPS-binding protein (LBP) in acute phase rabbit serum that enhanced the effect of LPS on macrophages in vitro and was essential for the inflammatory response to LPS in whole blood (8, 9). LBP is homologous with other phospholipid transport proteins and binds the lipid A moiety of LPS with 1:1 stoichiometry. LBP functions primarily as a phospholipid transport protein that disaggregates LPS and transfers it to targets on cellular membranes and to lipoproteins in solution. LBP facilitates the transport of LPS to high-density lipoproteins (HDL) and to experimental phospholipid monolayers in vitro (10, 11). LPS bound to HDL is biologically inactive, providing a mechanism to both transport and detoxify bacterial LPS in plasma (12, 13).

Studies with LPS-coated erythrocytes opsonized with LBP identified the CD14 differentiation marker on monocytes as a receptor for LBP, and antibodies to CD14 blocked LPS-dependent cellular activation in the blood (14). CD14 is anchored in the cellular membrane by a glycosyl-phosphatidyl-inositol (GPI) tail, and a soluble form lacking the GPI tail circulates in plasma. Soluble CD14 (sCD14) can accept LPS from LPS/LBP complexes and facilitate LPS-dependent activation of some CD14-negative cells (15). In addition, sCD14 participates with LBP in transferring LPS to HDL, inactivating LPS (16). The role of CD14 in the recognition of bacterial products has broadened, qualifying CD14 as a pattern-recognition receptor because inhibiting CD14 blocks or reduces the cellular responses to gram-positive bacteria and lipoarabinomannan from mycobacteria (3).

The crucial biologic roles of LBP and CD14 have been shown in studies with mice rendered deficient in either LBP or CD14 by targeted gene inactivation. Mice deficient in LBP did not mount inflammatory responses to small amounts of LPS or bacteria and did not die when challenged with LPS; however, they died more rapidly from a Salmonella infection (17). Serum from the LBP^/ mice could not support transfer of LPS to CD14, showing that no other serum proteins can substitute for LBP in this regard. Thus, the inflammatory response initiated by recognition of LPS is dependent on LBP, and this recognition response is critical for survival after a gram-negative bacterial challenge. Mice deficient in CD14 were resistant to LPS lethality, but unlike the LBP-deficient mice, they resisted bacterial challenge and had less bacteremia after a local infection than the control animals (18).

The structure of CD14 as a glycosyl/phosphatidyl/inositol-linked membrane protein, and transfection experiments showing that the membrane anchor of CD14 is not essential for cellular activation by CD14 (19), indicate that CD14 does not cause direct cellular activation by LPS, so the search has continued for a membrane protein in the LPS recognition pathway that can transmit intracellular signals. Independently, studies of the innate immune system in Drosophila identified Toll as a receptor that mediates dorsal-ventral development and antifungal defenses (20). The intracellular portion of Drosophila Toll has significant homology with the human interleukin (IL)-1 receptor, and a human Toll-like receptor (TLR) was cloned that mediated nuclear factor (NF)-B activation by IL-1-like signaling pathways (23). Genetic analysis of C3H/HeJ mice naturally resistant to LPS revealed a specific mutation in the intracellular portion of TLR4, providing direct biologic evidence of the critical role of TLR4 in LPS-dependent cellular activation (24). Humans carrying a mutation in the extracellular region of TLR4 are hyporesponsive to inhaled LPS (25). TLR4 and related proteins are now leading candidates as the critical membrane proteins that transmit signals from LPS via CD14.

The family of human TLRs now includes more than 10 members likely to mediate responses to different types of microbial stimuli (26). Although TLR2 was first found to confer LPS responsiveness in cell lines (27), more recent evidence shows that TLR2 mediates macrophage responses to yeast and gram-positive bacteria, whereas TLR4 facilitates recognition of gram-negative LPS (28, 29). Emerging evidence about tissue distribution of TLR forms indicates that TLR1 is widely expressed in tissues; TLR2, TLR4, and TLR5 are restricted to myeloid cells, and TLR3 is expressed only on dendritic cells (30, 31). Variations in TLR expression may explain cellular and tissue differences in the response to bacterial products.

	Recognition of Endotoxin in Normal and Inflamed Lungs

When bacterial endotoxin reaches the terminal airways and alveolar spaces, it encounters a lipid-rich environment in which surfactant lipids and surfactant-associated proteins line the thin, aqueous layer covering the epithelium. LBP and sCD14 are constituents of normal alveolar fluid, and membrane CD14 mediates the responses of alveolar macrophages to LPS in vitro (32). The concentrations of LBP and sCD14 in plasma and bronchoalveolar lavage fluid rise by more than ten-fold in patients with acute lung injury (33). Furthermore, in humans undergoing segmental antigen challenge in the lungs, the local concentration of LBP rises markedly within 24 h of allergen challenge (34). The sources of the LBP and sCD14 in lung fluids and the reasons for their dramatic rise in concentration in inflamed or injured lungs have been uncertain.

LBP is produced by hepatocytes as a type I acute phase response protein, and its production is stimulated by IL-6 and dexamethasone (35). LBP was not detected by the polymerase chain reaction in messenger RNA (mRNA) from the lungs of rabbits with a systemic acute phase response induced by subcutaneous turpentine, which induces LBP production in the liver (32). However, when the complementary DNA (cDNA) for rat LBP was cloned, Northern blot analysis of tissues from rats with acute phase reactions revealed an increased signal in the lung and kidney RNA, although the signal was much less intense than in liver RNA (39). Subsequently, Wong and coworkers showed that rat pulmonary artery smooth-muscle cells cultured in the presence of IL-1 produced an activity similar or identical to LBP (40). This established that LBP could be produced in the lungs, particularly in the setting of acute inflammation, but the biologic importance of LBP production by vascular smooth muscle cells required further study.

In this issue of the Red Journal, Dentener and associates provide the first evidence that LBP is produced by alveolar epithelial cells lining the air spaces of the lungs and is regulated by inflammatory cytokines produced at the onset of acute inflammatory responses (41). Initially, these investigators detected LBP mRNA in specimens of human lung RNA, providing a reason for investigating the source of LBP production in the lungs. LBP was detected by immunoassay in supernatants of three different types of cells: A549 cells, a malignant epithelial cell line derived from human lung adenocarcinoma; C10 cells, a nontransformed murine alveolar epithelial cell line; and primary isolates of human type II pneumocytes recovered from surgical lung specimens. LBP production was stimulated by the cytokines IL-1, TNF-, and IL-6, and by dexamethasoneall characteristic of LBP production by the liver. These findings provide support for the conclusion that LBP can be produced locally in the lungs, which may be particularly important in acute inflammatory responses such as ARDS, in which biologically active IL-1 and IL-6 are produced in the alveolar environment (42). A remaining question is how much LBP is actually produced locally in the lungs, particularly during inflammatory responses, and how much LBP derives from the microvascular circulation as a result of hepatic production.

The source of the sCD14 in normal and inflamed lung fluids is not completely clear. Like LBP, sCD14 circulates in the plasma, presumably shed from cell surface membranes like other GPI-anchored proteins. Movement of sCD14 from the plasma into the lungs is likely to explain some of the increase in sCD14 in alveolar fluids when permeability changes; however, local production and release in the lungs are also possible. Peripheral blood monocytes and alveolar macrophages release sCD14 during incubation, but blood monocytes bear considerably more membrane CD14 by flow cytometry and release more sCD14 during culture as compared with alveolar macrophages (45). IL-6 increases and IL-4 decreases sCD14 shedding from monocytes and alveolar macrophages. Extramyeloid expression of CD14 in lung tissue has been detected in mice treated with LPS or IL-1 (46, 47), providing additional sources of sCD14 at inflammatory foci. The biologic importance of the increased sCD14 in lung fluid needs to be clarified. Although Pugin and coworkers found that sCD14 can mediate activation of CD14-negative colonic epithelial cell lines in vitro, alveolar epithelial cells (A549) could not be activated by LPS/sCD14, and it is not clear whether normal distal airway epithelial cells can be activated by this mechanism (15).

The alveolar environment also contains other proteins and lipids that can bind LPS and modulate its biologic activity. The lipophilic nature of LPS suggests that it may partition in lung surfactant, although this has not yet been studied in detail. LPS both binds to and modulates the production of surfactant-associated proteins. LPS binds to SP-A in vitro via the lipid A moiety of LPS, which reduces the biologic activity of LPS in vitro (48). The concentrations of surfactant lipids and SP-A are high in normal lungs, but fall dramatically in injured lungs, reducing the potential for LPS binding and inactivation in acute lung injury (49, 50). The role of plasma lipoproteins that enter the lungs in modulating LPS bioactivity needs further study.

The current paradigm for the recognition of LPS in the air spaces suggests that LPS interactions vary as a function of the inflammatory environment and that LPS has very different biologic activities in normal and injured lungs (Figure 2). When LPS enters normal air spaces, it encounters high concentrations of SP-A, e.g., 250-500 µg/ml or higher, as estimated from measurements in diluted bronchoalveolar lavage fluid (50). In contrast, the concentrations of LBP are much lower, e.g., 50-100 ng/ml in undiluted alveolar fluid (33). Although the affinity of LBP for LPS is in the nanomolar range and is probably much greater than that of SP-A for LPS, the large excess of SP-A in normal air spaces is likely to minimize the biologic effects of low concentrations of LPS that enter the air spaces. In contrast, in patients with acute lung injury, in which IL-1, TNF-, and IL-6 all accumulate in the air spaces, the concentrations of SP-A and surfactant lipids fall to less than 10% of normal values (range of 25 µg/ml), whereas the concentration of LBP rises 100-fold or more (range of 5-10 µg/ml) (42, 49, 50). In this case, LBP is likely to be the dominant LPS-binding protein in the air spaces, amplifying the biologic effects of LPS in the lungs many-fold. These considerations are relevant not only for acute lung injury, but also for patients with airway inflammation, such as asthmatic patients exposed to inhaled allergens and LPS in the workplace (34). The allergic response increases local LBP concentrations, and the inflammatory responses to inhaled LPS may be dramatically enhanced.

View larger version (27K): [in this window] [in a new window]   Figure 2.   A simplified paradigm of LPS recognition in the air spaces. Right, LPS enters a normal space where the concentration of LBP is low and the concentration of SP-A is high. Binding to SP-A favors inactivation of LPS. Left, LPS enters an inflamed air space in which the concentration of LBP is increased by local production and exudation from plasma. In contrast, the concentration of SP-A is much lower than normal, due to the effects of IL-1 and other proinflammatory cytokines and possibly direct damage to Type II pneumocytes. Binding of LPS to LBP is favored by the much higher affinity of LBP for LPS (kD in the nanomolar range).

	Summary

Major advances have occurred in our understanding of the fundamental mechanisms that recognize bacterial products such as LPS and trigger innate immune responses. The lungs contain the largest surface area of the body in continuous contact with the outside environment, and studies of pulmonary responses provide an organ-specific relevance for basic studies in cellular systems. The study by Dentener and colleagues in this issue provides further evidence for the role of the LPS/LBP/CD14 pathway in the lungs by showing that LBP is a product of alveolar epithelial cells that is regulated by proinflammatory cytokines. Inhibition of this pathway may have therapeutic potential, as treatment of rabbits with a specific anti-CD14 monoclonal antibody protected them from repeated LPS challenge, even when the anti-CD14 antibody was administered after the first LPS challenge (51). Similarly, an anti-CD14 antibody protected primates from LPS-induced shock, improving hemodynamic parameters and minimizing circulating cytokine responses (52). In contrast, blocking CD14 in rabbits with pneumonia and sepsis improved systemic hemodynamics, but worsened gas exchange and delayed bacterial clearance from the lungs (53). The latter study suggests a dual role for the LPS/LBP/CD14 pathway: initiation of local innate immune defenses but mediation of deleterious systemic host responses when local infections are not cleared. The LPS/LBP/CD14 pathway provides new opportunities for targeting the host response to LPS and other bacterial products in the lungs, although caution is required to avoid altering the delicate balance leading to recognition and elimination of microbes from the lungs.

	Footnotes

Abbreviations: glycosyl-phosphatidyl-inositol, GPI; interleukin, IL; lipopolysaccharide-binding protein, LBP; lipopolysaccharide, LPS; soluble CD14, sCD14; Toll-like receptor, TLR.

(Received in original form June 6, 2000).

Acknowledgments: Supported in part by grants GM37696, HL 30542, and AI29103 from the National Institutes of Health and by the Medical Research Service of the U.S. Department of Veterans Affairs.

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