Citations

Altered natural killer (NK) cell function is a component of the global immune dysregulation that occurs in advanced malignancies. Another condition associated with altered NK homeostasis is normal pregnancy, where robust infiltration with CD16− CD9+ NK cells can be identified in decidual tissues, along with a concomitant expansion of CD16− NK cells in the maternal peripheral blood. In metastatic melanoma, we identified a similar expansion of peripheral blood CD16− NK cells (median 7.4% in 41 patients with melanoma compared with 3.0% in 29 controls,P<.001). A subset of NK cells in melanoma patients also expresses CD9, which is characteristically expressed only on NK cells within the female reproductive tract. Expansion of CD16− NK cells was associated with elevated plasma transforming growth factor-beta (TGF-βlevels (median 20 ng/ml, Spearman'sρ=0.81,P=.015)). These findings suggest the possibility of exploring anti-TGF-βtherapy to restore NK function in melanoma.

Abstract Thymic stromal lymphopoietin (TSLP) is a newly identified IL-7–like cytokine known to be expressed in airway biopsies of patients with asthma and chronic obstructive pulmonary disease. As both diseases may be induced or exacerbated by cigarette smoking, it is possible that TSLP represents an important link between cigarette smoke exposure and inflammatory signaling in the airways. In this regard, TSLP appears to also be expressed in airway smooth muscle (ASM); however, its role is unknown. In the current study, we examined TSLP and the TSLP receptor (TSLP-R) expression and function in human ASM cells under normal conditions and following exposure to cigarette smoke extract (CSE). Western blot analysis of human ASM cells showed significant expression of TSLP and TSLP-R, with increased expression of both by overnight exposure to 1 or 2% CSE. Furthermore, CSE increased TSLP release by ASM. In parallel experiments using enzymatically dissociated human ASM cells loaded with the Ca2+ indicator fura 2-AM and imaged using fluorescence microscopy, we evaluated the effects of CSE exposure on intracellular Ca2+ ([Ca2+]i) responses to agonist stimulation. [Ca2+]i responses to histamine were increased with overnight CSE exposure. Exposure to TSLP also resulted in elevated responses, which were blunted by TSLP and TSLP-R Abs. Importantly, the enhancing effects of CSE on [Ca2+]i responses were also blunted by these Abs. These effects were associated with CSE- and TSLP-induced changes in STAT5 phosphorylation. Overall, these novel data suggest that cigarette smoke, TSLP, and ASM are functionally linked and that cigarette smoke-induced increase in airway contractility may be mediated via ASM-derived increases in TSLP signaling.

The severity of asthma, a disease characterized by airway hyperresponsiveness and inflammation, is enhanced in some women during the menstrual cycle and during pregnancy but relieved in others. These clinical findings suggest that sex steroids modulate airway tone. Based on well-known relaxant effects of estrogens on vascular smooth muscle, we hypothesized that estrogens relax airway smooth muscle (ASM), thus facilitating bronchodilation. In ASM tissues from female patients, Western and immunocytochemical analyses confirmed the presence of both estrogen receptor (ER) isoforms, ERα and ERβ. In fura 2-loaded, dissociated ASM cells maintained in culture, acute exposure to physiological concentrations of 17β-estradiol (E2; 100 pM to 10 nM) decreased the intracellular Ca2+ ([Ca2+]i) response to 1 μM histamine, an effect reversed by the ER antagonist ICI-182,780. The ERα-selective agonist (R,R)-THC had a greater reducing effect on [Ca2+]i responses to histamine and 1 μM ACh compared with the ERβ-selective agonist (DPN). The effects of E2 on [Ca2+]i were mediated, at least in part, via decreased Ca2+ influx through l-type channels and store-operated Ca2+ entry but not via Ca2+-activated K+ channels, receptor-operated entry, or sarcoplasmic reticulum reuptake. Overall, these data support our hypothesis that estrogens relax ASM and suggest a potentially novel therapeutic target in airway hyperresponsiveness.

Abstract Purpose: Immunotherapeutic modalities are commonly used for treatment of patients with melanoma. The therapeutic success in preclinical models has not yielded the expected clinical results. To understand this discrepancy, we attempted to define immune homeostasis of 209 patients with melanoma across stages of disease relative to normal controls. Experimental Design: Peripheral blood mononuclear cells (PBMC) and plasma were collected from patients and healthy donors. PBMC were analyzed for frequencies of natural killer, dendritic, and T cells and their functional status. Matched plasma samples were analyzed for the concentrations of 27 cytokines, chemokines, and growth factors. RNA was isolated from 24 metastatic melanoma tumor biopsies and profiled by microarray analysis. Results: The frequency of natural killer, T, and dendritic cells in patients does not significantly change across stages of melanoma. However, plasma concentrations of Th2 cytokines [interleukin (IL)-4, IL-5, IL-10, and IL-13] in tumor-bearing patients were significantly higher than those with resected melanoma. Expression array analysis of metastatic melanoma revealed that the malignant melanocytes were not the source of the Th2 cytokines but did highly up-regulate vascular endothelial growth factor (VEGF) transcripts, consistent with plasma VEGF concentrations. In vitro VEGF exposure of normal PBMC lead to repolarization from Th1 to Th2 emulating the state of metastatic melanoma. Conclusions: Patients with metastatic melanoma exist in a state of Th2-mediated “chronic inflammation” as a result of at least VEGF overproduction by malignant tumors. These data support prior observations regarding the effect of VEGF on immune cell function and suggests consideration of VEGF inhibitors in future cancer immunotherapy clinical studies in metastatic melanoma.

Abstract There is increasing recognition that neurotrophin (NT) signaling occurs in non-neuronal tissues, including airway smooth muscle (ASM). We recently demonstrated that NTs, such as brain-derived neurotrophic factor (BDNF), enhance intracellular Ca2 + ([Ca2+]i) and force regulation in human ASM. Increased NT expression has been observed in airway diseases, such as asthma and allergy. In the present study, we tested the hypothesis that NTs contribute to inflammation-induced enhancement of ASM contractility. Using human ASM cells and real-time fluorescence [Ca2+]i imaging, we examined the contribution of the high-affinity tropomyosin-related kinase and low-affinity, pan-NT p75NTR receptors to [Ca2+]i regulation under control conditions and after exposure to the proinflammatory cytokine TNF-α (20 ng/ml). Exposure to TNF-α enhanced [Ca2+]i responses to agonist (acetylcholine, histamine). Exposure to 10 nM BDNF for even 30 minutes substantially and synergistically enhanced TNF-α effects on [Ca2+]i responses to agonist. Small interfering RNA suppression of tropomyosin-related kinase substantially blunted the effect of BDNF on [Ca2+]i responses to agonist (with greater effect on Ca2+ influx via store-operated Ca2+ entry compared with sarcoplasmic reticulum Ca2+ release) in both control and TNF-α–exposed cells. However, p75NTR suppression by small interfering RNA had no significant effect on [Ca2+]i responses in either cell group. These novel data demonstrate that NTs influence ASM contractility, and suggest a potential role for NTs in airway diseases.

Abstract Previous studies have suggested that the proinflammatory cytokine, TNF-α, contributes to airway hyperresponsivness by altering airway smooth muscle (ASM) Ca2+ responses to agonist stimulation. The present study examined the effects of TNF-α on Ca2+ influx pathways in cultured human ASM cells (HASMCs). Proteins encoded by the transient receptor potential (TRP) gene family function as channels through which receptor-operated and store-operated Ca2+ entry (SOCE) occur. In the present study, the presence of TRPC1, TRPC3, TRPC4, TRPC5, and TRPC6 mRNA and protein expression was confirmed in cultured HASMCs using RT-PCR and Western blot analysis. TNF-α treatment significantly increased TRPC3 mRNA and protein levels in HASMCs as well as SOCE. TNF-α treatment also increased both the peak and plateau intracellular Ca2+ concentration responses in HASMCs elicited by acetylcholine and bradykinin. The effects of TNF-α treatment on SOCE and agonist-induced intracellular Ca2+ concentration responses were attenuated using small interfering RNA transfection, which knocked down TRPC3 expression. Thus, in inflammatory airway diseases, TNF-α treatment may result in increased myocyte activation due to altered Ca2+ influx pathways. These results suggest that TRPC3 may be an important therapeutic target in inflammatory airway diseases such as asthma and chronic obstructive pulmonary disease.