The influence of Arhgef1 on pulmonary leukocyte function.

Resident leukocytes in the lungs of healthy individuals are necessary for the innate and adaptive immune response toward potentially harmful foreign antigens that are inhaled on a constant basis. Under normal circumstances, inflammatory stimuli are effectively eradicated via innate immunity with accompanying resolution of inflammation and repair of the lung tissue. Work in our lab has explored how Arhgef1, an intracellular signaling molecule expressed by hematopoietic cells, participates in immune function with an emphasis on its contribution to pulmonary immunity. This review summarizes our studies on the role of Arhgef1 in regulating pulmonary macrophage function.

Prostaglandin E2 deficiency uncovers a dominant role for thromboxane A2 in house dust mite-induced allergic pulmonary inflammation.

Prostaglandin E(2) (PGE(2)) is an abundant lipid inflammatory mediator with potent but incompletely understood anti-inflammatory actions in the lung. Deficient PGE(2) generation in the lung predisposes to airway hyperresponsiveness and aspirin intolerance in asthmatic individuals. PGE(2)-deficient ptges(-/-) mice develop exaggerated pulmonary eosinophilia and pulmonary arteriolar smooth-muscle hyperplasia compared with PGE(2)-sufficient controls when challenged intranasally with a house dust mite extract. We now demonstrate that both pulmonary eosinophilia and vascular remodeling in the setting of PGE(2) deficiency depend on thromboxane A(2) and signaling through the T prostanoid (TP) receptor. Deletion of TP receptors from ptges(-/-) mice reduces inflammation, vascular remodeling, cytokine generation, and airway reactivity to wild-type levels, with contributions from TP receptors localized to both hematopoietic cells and tissue. TP receptor signaling ex vivo is controlled heterologously by E prostanoid (EP)(1) and EP(2) receptor-dependent signaling pathways coupling to protein kinases C and A, respectively. TP-dependent up-regulation of intracellular adhesion molecule-1 expression is essential for the effects of PGE(2) deficiency. Thus, PGE(2) controls the strength of TP receptor signaling as a major bronchoprotective mechanism, carrying implications for the pathobiology and therapy of asthma.

Genetic variants of the receptors for thromboxane A2 and IL-4 in atopic dermatitis.

Thromboxane A2 (TXA2) is an arachidonate metabolite which is considered to relate to chronic inflammation in atopic diseases characterized by elevated immunoglobulin E productivity. The elevation of immunoglobulin E levels involves many molecules including interleukin-4 (IL-4) and interleukin-4 receptor alpha chain (IL-4R alpha). To assess whether genetic variants of TXA2 receptor, IL-4 and IL-4R alpha genes relate to the elevation of serum immunoglobulin E levels in patients with atopic dermatitis (AD), we conducted an association study of genetic polymorphisms of TXA2 receptor (795C/T), IL-4 (-589C/T), and IL-4R alpha (Ile50Val) in a Japanese population (n = 789). The TXA2 receptor 795TT genotype strongly related to AD with high serum immunoglobulin E concentrations. AD patients with both TXA2 receptor 795TT genotype and the IL-4R alpha Ile50/Ile50 genotype showed the greatest immunoglobulin E concentrations. These results suggest TXA2 receptor polymorphism strongly interacts with IL-4R alpha polymorphism as a major determinant of high serum immunoglobulin E levels in AD.

Inhibitory effect of a TP-receptor antagonist, S-1452, on antigen-induced nasal plasma exudation in guinea pig model for allergic rhinitis.

S-1452, a selective thromboxane (Tx) A(2) receptor (TP-receptor) antagonist, was evaluated in antigen- and U-46619 (a TxA(2) mimetic)-induced guinea pig nasal plasma exudation models. Exposure of the nasal cavity of actively sensitized guinea pigs to aerosolized ovalbumin (OA) caused marked exudation of dye into both the nasal mucosa and nasal airway lumen. These responses were significantly inhibited by S-1452 (30 mg/kg, p.o.) as well as an H(1)-antihistamine, diphenhydramine (5 mg/kg, i.v.). In addition, exposure of the nasal cavity of nonsensitized guinea pigs to aerosolized U-46619 or histamine also resulted in nasal plasma exudation, and S-1452 (1 mg/kg, p.o.) almost completely suppressed the U-46619-induced response but did not affect the histamine-induced one, even at a high dose of 30 mg/kg. These results indicate that TxA(2) as well as histamine may play an important role in antigen-induced nasal plasma exudation in guinea pigs, and S-1452 can be expected to be useful for the treatment of allergic rhinitis.

Down-regulation of T cell activation following inhibition of dipeptidyl peptidase IV/CD26 by the N-terminal part of the thromboxane A2 receptor.

Using synthetic inhibitors, it has been shown that the ectopeptidase dipeptidyl peptidase IV (DP IV) (CD26) plays an important role in the activation and proliferation of T lymphocytes. The human immunodeficiency virus-1 Tat protein, as well as the N-terminal nonapeptide Tat(1-9) and other peptides containing the N-terminal sequence XXP, also inhibit DP IV and therefore T cell activation. Studying the effect of amino acid exchanges in the N-terminal three positions of the Tat(1-9) sequence, we found that tryptophan in position 2 strongly improves DP IV inhibition. NMR spectroscopy and molecular modeling show that the effect of Trp(2)-Tat(1-9) could not be explained by significant alterations in the backbone structure and suggest that tryptophan enters favorable interactions with DP IV. Data base searches revealed the thromboxane A2 receptor (TXA2-R) as a membrane protein extracellularly exposing N-terminal MWP. TXA2-R is expressed within the immune system on antigen-presenting cells, namely monocytes. The N-terminal nonapeptide of TXA2-R, TXA2-R(1-9), inhibits DP IV and DNA synthesis and IL-2 production of tetanus toxoid-stimulated peripheral blood mononuclear cells. Moreover, TXA2-R(1-9) induces the production of the immunosuppressive cytokine transforming growth factor-beta1. These data suggest that the N-terminal part of TXA2-R is an endogenous inhibitory ligand of DP IV and may modulate T cell activation via DP IV/CD26 inhibition.

Localization of authentic thromboxane A2/prostaglandin H2 receptor in the rat kidney.

Using a polyclonal antibody against authentic thromboxane A2/prostaglandin H2 (TxA2/PGH2) receptor protein, we assessed the distribution of this receptor in the normal rat kidney by routine methods of immunofluorescence microscopy. The receptor localized both in glomeruli and in tubules. In the former, the distribution of the receptor was most prominent along the lumen of glomerular capillary loops. Parietal epithelial cells of the Bowman's capsule, podocytes and mesangial cells also demonstrated immunostainable receptor. In the tubules, the receptor localized most prominently at the base of the brush border of proximal tubules and at the luminal surface of thick ascending limbs and distal convoluted tubules. These observations point to sites that are likely to be targeted by thromboxane A2 in forms of renal injury characterized by enhanced synthesis of this eicosanoid.

Immunohistochemical localization of thromboxane receptor and thromboxane synthase in rat testis.

The cellular localization of thromboxane A2 receptor (TXR) and thromboxane synthase (TXS) in rat testes was examined with an antibody against the carboxyl-terminal tail of rat TXR and anti-porcine lung TXS antibody. By light microscopy immunoreactivity for TXR was shown to be present in spermatids, whereas spermatogonia, spermatocytes, and spermatozoa lacked the immunoreactivity. Immunoelectron microscopic analysis revealed that immunostainable TXR was present in acrosomes of spermatids. In contrast, immunoreactivity for TXS was present in all stages of spermatogenic cells; spermatogonia, spermatocytes, spermatids, and spermatozoa. TX system may possibly contribute to the formation of acrosomes or have some unrecognized functions in an autocrine/paracrine fashion.

Purification of rat brain, rabbit aorta, and human platelet thromboxane A2/prostaglandin H2 receptors by immunoaffinity chromatography employing anti-peptide and anti-receptor antibodies.

In the present study, a new polyclonal antibody (TxAb) was raised against native thromboxane A2 (TXA2)/prostaglandin H2 (PGH2) receptor protein. Previously developed anti-peptide antibodies (P1Ab, P2Ab) and TxAb were then used to prepare immunoaffinity columns to purify TXA2/PGH2 receptors from platelets, brain, and aorta. In platelets, SDS-polyacrylamide gel electrophoresis revealed the purification of a 55-kDa protein by each affinity column. Identification of this protein as the TXA2/PGH2 receptor was based on: 1) an identical electrophoretic mobility to authentic receptor; 2) immunoblotting of TxAb against P1Ab and P2Ab-purified protein; 3) immunoblotting of P1Ab/P2Ab against TxAb-purified protein; and 4) specific [3H]SQ29,548 binding to TxAb-purified protein. P1Ab/TxAb purification of receptors from brain revealed a major protein band at 55 kDa. Furthermore, the eluates from ligand affinity chromatography confirmed the presence of this 55-kDa protein in brain (which was immunoblotted with TxAb), and contained specific [3H]SQ29,548 binding. In addition to the 55-kDa protein, P1Ab/TxAb also purified a minor protein in brain at 52 kDa, which when concentrated, cross-blotted with TxAb and P1Ab. This finding indicates sequence homology between the 55- and 52-kDa proteins. Independent identification of brain TXA2/PGH2 receptors was provided by P2Ab/TxAb immunohistochemistry, which demonstrated specific labeling of discrete myelin-containing fiber tracts. P2Ab/TxAb purification of TXA2/PGH2 receptors from aorta also revealed a major protein band at 55 kDa and a minor band at 52 kDa. These results represent the first purification of TXA2/PGH2 receptors from either brain or aorta.

Anti-peptide antibodies against the human blood platelet thromboxane A2/prostaglandin H2 receptor. Production, purification and characterization.

Two anti-peptide antibodies have been raised against the human blood platelet thromboxane A2/prostaglandin H2 (TXA2/PGH2) receptor. Based on the published sequence of the placental TXA2/PGH2 receptor, two decapeptide segments were selected as potential antigens: one in the first extracellular loop corresponding to residue 89 through 98, and the other in the C-terminal region of the intracellular domain corresponding to residue 314 through 323. Rabbits were immunized with each peptide, and the antisera were subjected to a two-step purification procedure. The IgG fraction was purified using a DEAE Affi-Gel Blue column, and the peptide-specific IgG was further purified by affinity chromatography employing each peptide as the immobilized ligand. The combined purification factor for both procedures was approximately 60-fold. By ELISA, both antibodies displayed immunoreactivity toward their synthetic antigens, solubilized platelet membranes and affinity-purified TXA2/PGH2 receptor protein. Furthermore, Western blot analysis revealed that: (1) each antibody reacted with the purified platelet TXA2/PGH2 receptor protein (55 kDa); and (2) each antibody recognized a single band (55 kDa) in solubilized platelet membranes. These findings establish antibody specificity for the human platelet TXA2/PGH2 receptor protein. Functional analysis demonstrated that neither antibody interfered with ADP- or U46619-induced platelet aggregation of [3H]SQ29,548 binding to the solubilized receptor. These results suggest that the antibody epitopes are separate from the TXA2/PGH2 binding domain. In summary, two specific anti-peptide antibodies have been raised against the human platelet TXA2/PGH2 receptor. These antibodies should prove to be of value in the further investigation of the platelet TXA2/PGH2 receptor.

Monoclonal anti-idiotypic antibody to a potent thromboxane A2 receptor antagonist and its interaction with thromboxane A2 receptor.

A monoclonal anti-idiotypic antibody that interacts with thromboxane A2 receptor was generated using an anti-idiotypic approach. Idiotypic antibodies against a potent receptor antagonist, HS-145, were generated in rabbit. The idiotypic antibodies were then selected by an affinity procedure using SQ29,548-Affi-Gel-102 matrix. The selected idiotypic antibodies were used as surrogate receptor for anti-idiotypic antibody generation. A mouse monoclonal antibody, 3D-9E-12, was generated. It was shown to displace 125I-HS-145 from affinity-purified idiotypic antibodies. It also inhibits 125I-IS-145 binding to thromboxane A2 receptor in human platelet membranes in a dose-dependent manner. Furthermore, it attenuated U46,619-induced increase in [35S]guanosine 5'-O-(thiotriphosphate) binding and GTPase activity in human platelet membranes. Finally, it inhibited U46,619- but not PAF-induced platelet aggregation. These results indicate that 3D-9E-12 acts as a specific antagonist in the thromboxane A2 receptor.