The Hemostatic Mechanism and Allergy

Toll-like receptor 4 (TLR4) is most commonly known as a receptor for lipopolysaccharide (LPS, endotoxin). Indeed, the gene encoding TLR4 was identified by positional cloning using LPS-resistant C3H/HeJ mice.¹ Subsequently, many ligands for TLR4 have been identified, including taxol, heparan sulfate, and hyaluronate.²  TLR4 is expressed by several types of cells, including monocytes, macrophages, dendritic cells, mast cells, and B cells. Ligation of TLR4 initiates signaling via MyD88- and TRIF-dependent pathways, which leads to activation of innate immune system functions. The innate immune system is an ancient set of mechanisms brought into play by several classes of TLRs in response to microbial infection. While necessary for control of specific microbial agents, as witnessed by susceptibility to infection associated with genetic defects in TLR-dependent pathways, the flip side of TLR activity is involvement in autoimmunity and allergy. For example, TLR4 is involved in Type 2 T-helper cell (T_H2)-dependent allergic IgE responses, including asthma and other airway disorders.

Allergic responses to pollen, dust mite antigens, and fungi are associated with proteinase-dependent and proteinase-independent T_H2 cell-mediated events. To study the role of TLR4 in allergic inflammation, Millien et al. in the laboratory of David Corry, Baylor College of Medicine, Houston, Texas, subjected mice to intranasal challenge with several known allergens, including a proteinase derived from Aspergillus, live conidia (spores) from Aspergillus niger, and ovalbumin. Wild-type (wt) mice developed airway hyper-responsiveness, eosinophilia, and other findings consistent with allergic asthma when exposed to the immunogens. These responses were significantly attenuated in TLR4^–/–mice. Consistent with its dual role in allergy and immunity, TLR4^–/– mice were also defective in clearing A. niger following inhalational challenge. In contrast, there was no difference between wt and TLR4^–/– mice in T_H2-dependent pulmonary IL-4 secretion and IgE levels. This result indicates that TLR4 does not produce a T_H2-dependent process, but rather is involved in the response to a T_H2-mediated process.

Exposure of murine bone marrow-derived macrophages (BMDMs) or alveolar macrophages to LPS, or fungal proteinase, resulted in upregulation of several genes associated with antifungal immunity, including lysozyme, Marco, and secretory leukoproteinase inhibitor. Fungal proteinase-activated, but not naïve, BMDMs inhibited fungal growth when A. niger conidia were added to cell cultures. This fungistatic effect required the presence of serum-containing media. Because fibrinogen has been identified as a TLR4 ligand,³  the authors examined the possible role of fibrinogen-derived polypeptides as the putative serum factors. They found that thrombin could substitute for fungal proteinase, indicating that fibrin formation is involved in serum-dependent fungistasis. They produced fibrinogen cleavage products (FCPs) by adding thrombin to purified fibrinogen and found that FCPs could substitute for fungal protease and serum as a fungistatic agent. FCPs also induced the expression of mRNA for IL-13Rα1 and the airway mucin gene Muc5ac, which are components of allergic responses, from BMDMs. In contrast, when TLR4^–/– BMDMs were used, FCPs or the combination of fungal proteinase and serum did not produce fungistasis. Intranasal challenge of mice with FCPs did not produce airway hyperresponsiveness or pulmonary IL-4 secretion, consistent with the hypothesis that TLR4 mediates, but does not drive, T_H2-dependent responses. However, hirudin, a specific thrombin inhibitor, attenuated both fungal proteinase and ovalbumin-mediated experimental asthma.