K. Frank Austen

Significance: Leukotriene E₄(LTE₄), a lipid mediator detected in asthma exacerbations triggered by allergen, viruses, and aspirin, elicits airflow obstruction and lung inflammation in asthmatics. GPR99 is the recently identified high-affinity receptor for LTE₄and is resistant to blockade by commercially available cysteinyl leukotriene (cysLT) receptor antagonists. Here we find that GPR99 is expressed in respiratory epithelial cells and mediates mucin release and submucosal swelling in response to LTE₄or to cysLTs elicited byAlternaria, a common airborne fungus. Furthermore, among cysLT receptors, only GPR99 regulates baseline numbers of mucin-containing goblet cells. These studies demonstrate a unique role for GPR99 in epithelial cell homeostasis and activation and indicate that epithelial cells may be a dominant site of LTE₄action in the lung.

Atopic dermatitis (AD) skin lesions exhibit epidermal and dermal thickening, eosinophil infiltration, and increased levels of the cysteinyl leukotriene (cys-LT) leukotriene C₄(LTC₄). Epicutaneous sensitization with ovalbumin of WT mice but not ΔdblGATA mice, the latter of which lack eosinophils, caused skin thickening, collagen deposition, and increased mRNA expression of the cys-LT generating enzyme LTC₄synthase (LTC₄S). Skin thickening and collagen deposition were significantly reduced in ovalbumin-sensitized skin of LTC₄S-deficient and type 2 cys-LT receptor (CysLT₂R)–deficient mice but not type 1 cys-LT receptor (CysLT₁R)-deficient mice. Adoptive transfer of bone marrow-derived eosinophils from WT but not LTC₄S-deficient mice restored skin thickening and collagen deposition in epicutaneous-sensitized skin of ΔdblGATA recipients. LTC₄stimulation caused increased collagen synthesis by human skin fibroblasts, which was blocked by CysLT₂R antagonism but not CysLT₁R antagonism. Furthermore, LTC₄stimulated skin fibroblasts to secrete factors that elicit keratinocyte proliferation. These findings establish a role for eosinophil-derived cys-LTs and the CysLT₂R in the hyperkeratosis and fibrosis of allergic skin inflammation. Strategies that block eosinophil infiltration, cys-LT production, or the CysLT₂R might be useful in the treatment of AD.

Mouse mast cells (MCs) express a large number of serine proteases including tryptases, mouse mast cell protease (mMCP)-6 and -7; chymases, mMCP-1, -2, and -4; and an elastase, mMCP-5; along with carboxypeptidase-A3 (CPA3). In helminth-infected mouse intestine, distinct protease phenotypes are observed for connective tissue MCs (CTMCs) (mMCP-4⁺–7⁺, and CPA3⁺) and mucosal MCs (MMCs) (mMCP-1⁺and 2⁺). To determine whether the protease phenotype was regulated by the tissue, we compared the phenotype of constitutive CTMCs and induced MMCs in trachea and large airways in antigen-sensitized unchallenged and challenged mice to MCs in skin and helminthic-infected intestine. We found that in the trachea, unlike in skin and intestine, CTMCs and MMCs both express all six serine proteases and CPA3 (mMCP-1⁺, -2⁺, 4⁺–7⁺, CPA3⁺). This phenotype also holds for the lung CTMCs in the proximal bronchi, whereas the induced MMCs express only four proteases, mMCP-1, -2, -6, and -7. Thus, the T-cell–dependent induction of MMCs in trachea, large bronchi, and small intestine provides numbers but does not determine the protease phenotype. Furthermore, the CTMCs, which are constitutive, also show striking differences at these tissue sites, supporting the view that the differences in expression are tissue directed and not dependent on inflammation.

The cysteinyl leukotrienes (cys-LTs) are proinflammatory lipid mediators acting on the type 1 cys-LT receptor (CysLT ₁ R) to mediate smooth muscle constriction and vascular permeability. GPR17, a G protein-coupled orphan receptor with homology to the P2Y and cys-LT receptors, failed to mediate calcium flux in response to leukotriene (LT) D ₄ with stable transfectants. However, in stable cotransfections of 6×His-tagged GPR17 with Myc-tagged CysLT ₁ R, the robust CysLT ₁ R-mediated calcium response to LTD ₄ was abolished. The membrane expression of the CysLT ₁ R analyzed by FACS with anti-Myc Ab was not reduced by the cotransfection, yet both LTD ₄ -elicited ERK phosphorylation and the specific binding of [ ³ H]LTD ₄ to microsomal membranes were fully inhibited. CysLT ₁ R and GPR17 expressed in transfected cells were coimmunoprecipitated and identified by Western blots, and confocal immunofluorescence microscopy revealed that GPR17 and CysLT ₁ R colocalize on the cell surface of human peripheral blood monocytes. Lentiviral knockdown of GPR17 in mouse bone marrow-derived macrophages (BMMΦs) increased both the membrane expression of CysLT ₁ R protein by FACS analysis and the LTD ₄ -elicited calcium flux in a dose-dependent manner as compared with control BMMΦs, indicating a negative regulatory function of GPR17 for CysLT ₁ R in a primary cell. In IgE-dependent passive cutaneous anaphylaxis, GPR17-deficient mice showed a marked and significant increase in vascular permeability as compared with WT littermates, and this vascular leak was significantly blocked by pretreatment of the mice with the CysLT ₁ R antagonist, MK-571. Taken together, our findings suggest that GPR17 is a ligand-independent, constitutive negative regulator for the CysLT ₁ R that suppresses CysLT ₁ R-mediated function at the cell membrane.

The cysteinyl leukotrienes (cys-LTs) are a family of potent lipid mediators of inflammation derived from arachidonic acid. Activation of certain cell types results in the biosynthesis and export of leukotriene (LT) C₄, which then undergoes extracellular metabolism to LTD₄and LTE₄. LTE₄, the most stable cys-LT, is only a weak agonist for the defined type 1 and type 2 cys-LT receptors (CysLT₁R and CysLT₂R, respectively). We had recognized a greater potency for LTE₄than LTC₄or LTD₄in constricting guinea pig tracheain vitroand comparable activity in eliciting a cutaneous wheal and flare response in humans. Thus, we hypothesized that a vascular permeability response to LTE₄in mice lacking both the CysLT₁R and CysLT₂R could establish the existence of a separate LTE₄receptor. We now report that the intradermal injection of LTE₄into the ear of mice deficient in both CysLT₁R and CysLT₂R elicits a vascular leak that exceeds the response to intradermal injection of LTC₄or LTD₄, and that this response is inhibited by pretreatment of the mice with pertussis toxin or a Rho kinase inhibitor. LTE₄is ≈64-fold more potent in the CysLT₁R/CysLT₂R double-deficient mice than in sufficient mice. The administration of a CysLT₁R antagonist augmented the permeability response of the CysLT₁R/CysLT₂R double-deficient mice to LTC₄, LTD₄, and LTE₄. Our findings establish the existence of a third receptor, CysLT_ER, that responds preferentially to LTE₄, the most abundant cys-LT in biologic fluids, and thus reveal a new target for therapeutic intervention.

I have spent my entire professional life at Harvard Medical School, beginning as a medical student. I have enjoyed each day of a diverse career in four medical subspecialties while following the same triad of preclinical areas of investigation—cysteinyl leukotrienes, mast cells, and complement—with occasional translational opportunities. I did not envision a career with a predominant preclinical component. Such a path simply evolved because I chose instinctively at multiple junctures to follow what proved to be propitious opportunities. My commentary notes some of the highlights for each area of interest and the mentors, collaborators, and trainees whose counsel has been immensely important at particular intervals or over an extended period.

Cysteinyl leukotrienes are established mediators of bronchial asthma and have agonist roles analogous to those of histamine in allergic rhinitis. We now know that the substance originally termed slow-reacting substance of anaphylaxis was composed of three cysteinyl leukotrienes that act in the inflammatory response via receptors on smooth muscle and on bone marrow–derived inflammatory cells. K. Frank Austen describes the work culminating in the identification, biosynthesis and functional characterization of these moieties.

Chemokine receptors regulate the trafficking of leukocytes by mediating chemotaxis and by their influence on the expression and/or affinity of leukocyte integrins. Using blocking mAb, we showed that antigen-induced recruitment of mast cell progenitors (MCp) to the lung requires interaction of a4 integrins on the MCp with endothelial vascular cell adhesion molecule 1 (VCAM-1). In seeking a chemokine component, we found that CXCR2-deficient but not CCR3- or CCR5-deficient sensitized and antigen-challenged mice have significantly fewer lung MCp 1 day after challenge and fewer tracheal intraepithelial MC 1 week after challenge, implying that recruited MCp provide the source for these mature MC. Unexpectedly, reconstitution of sensitized, sublethally irradiated +/+ and −/− mice with bone marrow cells of either genotype indicated that expression of CXCR2 by the migrating MCp was not required. Instead, receptor function by resident lung cells was required because normal BM did not reconstitute MCp recruitment in irradiated CXCR2^−/−mice. The reduced MCp influx into the lung of CXCR2^−/−mice was accompanied by reduced induction of VCAM-1 transcripts and reduced endothelial surface expression. Thus, these studies demonstrate a role for a chemokine receptor in regulating endothelial VCAM-1 expression, MCp migration, and the level of intraepithelial MC in the lung of aerosolized, antigen-challenged mice.

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