PTP1B deficiency exacerbates inflammation and accelerates leukocyte trafficking in vivo.

It is reported that PTP1B limits cytokine signaling in vitro. However, PTP1B's function during inflammation in vivo is not known. In this report, we determined whether PTP1B deficiency affects allergic inflammation in vivo. Briefly, lungs of OVA-challenged PTP1B(-/-) mice had elevated numbers of eosinophils and eosinophil progenitors at 6 h after one OVA challenge and at 24 h after a third OVA challenge as compared with OVA-challenged wild-type mice. There was also an increase in numbers of CD11b(+)SiglecF(+)CD34(+)IL-5Rα(+) eosinophil progenitors in the bone marrow, peripheral blood, and spleens of OVA-challenged PTP1B(-/-) mice. Intravital microscopy revealed that, in OVA-challenged PTP1B(-/-) mice, blood leukocytes rapidly bound to endothelium (5-30 min), whereas, in wild-type mice, blood leukocytes bound to endothelium at the expected 6-18 h. Consistent with early recruitment of leukocytes, lung eotaxin and Th2 cytokine levels were elevated early in the PTP1B(-/-) mice. Interestingly, spleen leukocytes from PTP1B(-/-) mice exhibited an increased chemotaxis, chemokinesis, and transendothelial migration in vitro. In summary, PTP1B functions as a critical negative regulator to limit allergic responses.

Supplemental and highly elevated tocopherol doses differentially regulate allergic inflammation: reversibility of α-tocopherol and γ-tocopherol's effects.

We have reported that supplemental doses of the α- and γ-tocopherol isoforms of vitamin E decrease and increase, respectively, allergic lung inflammation. We have now assessed whether these effects of tocopherols are reversible. For these studies, mice were treated with Ag and supplemental tocopherols in a first phase of treatment followed by a 4-wk clearance phase, and then the mice received a second phase of Ag and tocopherol treatments. The proinflammatory effects of supplemental levels of γ-tocopherol in phase 1 were only partially reversed by supplemental α-tocopherol in phase 2, but were completely reversed by raising α-tocopherol levels 10-fold in phase 2. When γ-tocopherol levels were increased 10-fold (highly elevated tocopherol) so that the lung tissue γ-tocopherol levels were equal to the lung tissue levels of supplemental α-tocopherol, γ-tocopherol reduced leukocyte numbers in the lung lavage fluid. In contrast to the lung lavage fluid, highly elevated levels of γ-tocopherol increased inflammation in the lung tissue. These regulatory effects of highly elevated tocopherols on tissue inflammation and lung lavage fluid were reversible in a second phase of Ag challenge without tocopherols. In summary, the proinflammatory effects of supplemental γ-tocopherol on lung inflammation were partially reversed by supplemental levels of α-tocopherol but were completely reversed by highly elevated levels of α-tocopherol. Also, highly elevated levels of γ-tocopherol were inhibitory and reversible in lung lavage but, importantly, were proinflammatory in lung tissue sections. These results have implications for future studies with tocopherols and provide a new context in which to review vitamin E studies in the literature.

Vitamin E isoforms directly bind PKCα and differentially regulate activation of PKCα.

Vitamin E isoforms have opposing regulatory effects on leucocyte recruitment during inflammation. Furthermore, in vitro, vitamin E isoforms have opposing effects on leucocyte migration across endothelial cells by regulating VCAM (vascular cell-adhesion molecule)-1 activation of endothelial cell PKCα (protein kinase Cα). However, it is not known whether tocopherols directly regulate cofactor-dependent or oxidative activation of PKCα. We report in the present paper that cofactor-dependent activation of recombinant PKCα was increased by γ-tocopherol and was inhibited by α-tocopherol. Oxidative activation of PKCα was inhibited by α-tocopherol at a 10-fold lower concentration than γ-tocopherol. In binding studies, NBD (7-nitrobenz-2-oxa-1,3-diazole)-tagged α-tocopherol directly bound to full-length PKCα or the PKCα-C1a domain, but not PKCζ. NBD-tagged α-tocopherol binding to PKCα or the PKCα-C1a domain was blocked by diacylglycerol, α-tocopherol, γ-tocopherol and retinol, but not by cholesterol or PS (phosphatidylserine). Tocopherols enhanced PKCα-C2 domain binding to PS-containing lipid vesicles. In contrast, the PKCα-C2 domain did not bind to lipid vesicles containing tocopherol without PS. The PKCα-C1b domain did not bind to vesicles containing tocopherol and PS. In summary, α-tocopherol and γ-tocopherol bind the diacylglycerol-binding site on PKCα-C1a and can enhance PKCα-C2 binding to PS-containing vesicles. Thus the tocopherols can function as agonists or antagonists for differential regulation of PKCα.

Inhibition of allergic inflammation by supplementation with 5-hydroxytryptophan.

Clinical reports indicate that patients with allergy/asthma commonly have associated symptoms of anxiety/depression. Anxiety/depression can be reduced by 5-hydroxytryptophan (5-HTP) supplementation. However, it is not known whether 5-HTP reduces allergic inflammation. Therefore, we determined whether 5-HTP supplementation reduces allergic inflammation. We also determined whether 5-HTP decreases passage of leukocytes through the endothelial barrier by regulating endothelial cell function. For these studies, C57BL/6 mice were supplemented with 5-HTP, treated with ovalbumin fraction V (OVA), house dust mite (HDM) extract, or IL-4, and examined for allergic lung inflammation and OVA-induced airway responsiveness. To determine whether 5-HTP reduces leukocyte or eosinophil transendothelial migration, endothelial cells were pretreated with 5-HTP, washed and then used in an in vitro transendothelial migration assay under laminar flow. Interestingly, 5-HTP reduced allergic lung inflammation by 70-90% and reduced antigen-induced airway responsiveness without affecting body weight, blood eosinophils, cytokines, or chemokines. 5-HTP reduced allergen-induced transglutaminase 2 (TG2) expression and serotonylation (serotonin conjugation to proteins) in lung endothelial cells. Consistent with the regulation of endothelial serotonylation in vivo, in vitro pretreatment of endothelial cells with 5-HTP reduced TNF-α-induced endothelial cell serotonylation and reduced leukocyte transendothelial migration. Furthermore, eosinophil and leukocyte transendothelial migration was reduced by inhibitors of transglutaminase and by inhibition of endothelial cell serotonin synthesis, suggesting that endothelial cell serotonylation is key for leukocyte transendothelial migration. In summary, 5-HTP supplementation inhibits endothelial serotonylation, leukocyte recruitment, and allergic inflammation. These data identify novel potential targets for intervention in allergy/asthma.

Isoforms of vitamin E differentially regulate inflammation.

Vitamin E regulation of disease has been extensively studied in humans, animal models and cell systems. Most of these studies focus on the α-tocopherol isoform of vitamin E. These reports indicate contradictory outcomes for anti-inflammatory functions of the α-tocopherol isoform of vitamin E, especially with regards to clinical studies of asthma and atherosclerosis. These seemingly disparate clinical results are consistent with recently reported unrecognized properties of isoforms of vitamin E. Recently, it has been reported that physiological levels of purified natural forms of vitamin E have opposing regulatory functions during inflammation. These opposing regulatory functions by physiological levels of vitamin E isoforms impact interpretations of previous studies on vitamin E. Moreover, additional recent studies also indicate that the effects of vitamin E isoforms on inflammation are only partially reversible using physiological levels of a vitamin E isoform with opposing immunoregulatory function. Thus, this further influences interpretations of previous studies with vitamin E in which there was inflammation and substantial vitamin E isoforms present before the initiation of the study. In summary, this review will discuss regulation of inflammation by vitamin E, including alternative interpretations of previous studies in the literature with regards to vitamin E isoforms.