Toll-like receptor 7 rapidly relaxes human airways.

RATIONALE: Toll-like receptors (TLRs) 7 and 8 detect respiratory virus single-stranded RNA and trigger an innate immune response. We recently described rapid TLR7-mediated bronchodilation in guinea pigs. OBJECTIVES: To characterize TLR7 expression and TLR7-induced airway relaxation in humans and in eosinophilic airway inflammation in guinea pigs. To evaluate the relaxant effects of other TLRs. METHODS: Human airway smooth muscle strips were contracted with methacholine in vitro, and responses to TLR7 and TLR8 agonists were assessed. TLR7-mediated nitric oxide production was measured using a fluorescent indicator, and TLR7 expression was characterized using immunofluorescence. TLR7 signaling was also evaluated in ovalbumin-challenged guinea pigs. MEASUREMENTS AND MAIN RESULTS: The TLR7 agonist imiquimod (R837) caused rapid dose-dependent relaxation of methacholine-contracted human airways in vitro. This was blocked by the TLR7 antagonist IRS661 and by inhibiting nitric oxide production but not by inhibiting prostaglandin production. TLR7 activation markedly increased fluorescence of a nitric oxide detector. TLR7 was expressed on airway nerves, but not airway smooth muscle, implicating airway nerves as the source of TLR7-induced nitric oxide production. TLR7-mediated relaxation persisted in inflamed guinea pigs airways in vivo. The TLR8 agonists polyuridylic acid and polyadenylic acid also relaxed human airways, and this was not blocked by the TLR7 antagonist or by blocking nitric oxide or prostaglandin production. No other TLRs relaxed the airways. CONCLUSIONS: TLR7 is expressed on airway nerves and mediates relaxation of human and animal airways through nitric oxide production. TLR7-mediated bronchodilation may be a new therapeutic strategy in asthma.

Progressive muscle relaxation and secretory immunoglobulin A.

30 healthy students produced saliva samples for Immunoglobulin A assay before and after sessions involving either progressive muscle relaxation (n = 15) or a control condition (n = 15). Levels of immunoglobulin A increased significantly in the relaxation group but not in the control group.

Bi-directional activation between human airway smooth muscle cells and T lymphocytes: role in induction of altered airway responsiveness.

Because both T lymphocyte and airway smooth muscle (ASM) cell activation are events fundamentally implicated in the pathobiology of asthma, this study tested the hypothesis that cooperative intercellular signaling between activated T cells and ASM cells mediates proasthmatic changes in ASM responsiveness. Contrasting the lack of effect of resting human T cells, anti-CD3-activated T cells were found to adhere to the surface of naive human ASM cells, increase ASM CD25 cell surface expression, and induce increased constrictor responsiveness to acetylcholine and impaired relaxation responsiveness to isoproterenol in isolated rabbit ASM tissues. Comparably, exposure of resting T cells to ASM cells prestimulated with IgE immune complexes reciprocally elicited T cell adhesion to ASM cells and up-regulated T cell expression of CD25. Extended studies demonstrated that: 1) ASM cells express mRNAs and proteins for the cell adhesion molecules (CAMs)/costimulatory molecules, CD40, CD40L, CD80, CD86, ICAM-1 (CD54), and LFA-1 (CD11a/CD18); 2) apart from LFA-1, ASM cell surface expression of the latter molecules is up-regulated in the presence of activated T cells; and 3) pretreatment of ASM cells and tissues with mAbs directed either against CD11a or the combination of CD40 and CD86 completely abrogated both the activated T cell-induced changes in expression of the above CAMs/costimulatory molecules in ASM cells and altered ASM tissue responsiveness. Collectively, these observations identify the presence of bi-directional cross-talk between activated T cells and ASM cells that involves coligation of specific CAMs/costimulatory molecules, and this cooperative intercellular signaling mediates the induction of proasthmatic-like changes in ASM responsiveness.

Leukocyte integrin very late antigen-4/vascular cell adhesion molecule-1 adhesion pathway in splanchnic artery occlusion shock.

We investigated the role played by the very late antigen-4 (VLA-4)/ vascular cell adhesion molecule-1 (VCAM-1) interaction in the pathogenesis of splanchnic artery occlusion shock. Splanchnic artery occlusion shock was induced in anaesthetized rats by clamping splanchnic arteries for 45 min. Sham operated animals were used as controls. Survival time, serum tumour necrosis factor (TNF-alpha), monocyte and lymphocyte cell count and the responsiveness to acetylcholine of aortic rings were studied. Furthermore we investigated the VCAM-1 expression on vessel endothelium and the percentage of VLA-4 positive leukocytes. Splanchnic artery occlusion shocked rats had a decreased survival time (76 +/- 10 min, while sham shocked rats survived more than 4 h), increased serum levels of TNF-alpha (328 +/- 11 U/ml), a decreased number of both monocytes and lymphocytes and reduced responsiveness to acetylcholine (10 nM-10 microM) of aortic rings. In addition we found an increased expression of endothelial VCAM-1 on aortic rings and a reduced percentage of VLA-4 positive lymphocytes and monocytes. Passive immunization with specific antibodies raised against either VCAM-1 or VLA-4 (2 mg/kg, i.v., 3 h before splanchnic artery occlusion shock) increased survival, improved monocyte and lymphocyte count and restored the responsiveness of aortic rings to acetylcholine (P < 0.01). Finally, inhibition of TNF-alpha biosynthesis reversed the increased endothelial expression of VCAM-1 and the reduced percentage of integrin VLA-4 positive leukocytes. Our findings suggest that (i) VLA-4/VCAM-1 interaction has a role in the pathogenesis of circulatory shock; (ii) this interaction might be a target for new therapeutic approaches to the therapy of low-flow states.

Sensitization decreases relaxation in human isolated airways.

Passively sensitized human isolated airways provide an opportunity to study some aspects of bronchial hyperresponsiveness in vitro. Since it has been suggested that excessive airway narrowing could be due to impaired relaxation, we examined the effect of a variety of agents producing relaxation via different mechanisms, i.e., verapamil and lemakalim (a calcium channel antagonist and a potassium channel opener, respectively) and isoproterenol, forskolin, and dibutyryl cAMP (modulators of the beta-adrenoceptor signal transduction pathway). Human bronchial rings, obtained at thoracotomy, were passively sensitized by incubation in serum from atopic asthmatic patients, and control rings were incubated in serum from nonatopic subjects. We also studied bronchial rings from five spontaneously sensitized human lung specimens. Responses to the relaxant compounds were measured isometrically. Passive sensitization significantly decreased the efficacy of verapamil in maximally contracted tissues from 60 +/- 10 to 45 +/- 7% of the maximal carbachol response (n = 6, p < 0.05) and that of lemakalim from 51 +/- 16 to 38 +/- 14% (n = 7, p < 0.05) in tissues at baseline tone. Similarly, spontaneously sensitized tissues relaxed less to lemakalim (64 +/- 6% of the maximal response to isoproterenol, n = 5, p < 0.05) than did nonsensitized tissues (80 +/- 4%). Sensitization did not alter responses to isoproterenol, forskolin, and dibutyryl cAMP. We conclude that sensitization of human isolated airways reduces relaxation responses that depend upon activation of ion channels but not those that depend upon activation of beta-adrenoceptors and transduction processes directly coupled to these receptors.

Use of a new index to study relaxation in a vascular model of anaphylactic shock.

We have reported increased smooth muscle shortening ability in ragweed pollen-sensitized saphenous vein (SSV). This may account for the vascular hyperreactivity of anaphylactic shock. We have now investigated relaxation in SSV. Because isotonic relaxation is load and initial contractile element length dependent, we developed an adjusted half-relaxation time index, which was independent of these variables. Muscle activation state was monitored by measuring maximum unloaded velocity. The relaxation index showed no difference between SSV and control saphenous vein after 2.5, 10, and 15 s of electrical stimulation; however, after 1 s of stimulation it was prolonged significantly in SSV. We concluded that the cross bridges activating early in contraction demonstrated prolonged relaxation. Activation state during muscle relaxation spontaneously increased toward the end of relaxation, coincident with a slowing in isotonic re-elongation rate. This was seen only in muscles relaxing from 15 s of stimulation. Our results indicate that 1) the relaxation properties of early cycling (1 s) cross bridges are altered after sensitization; and 2) toward the end of isotonic relaxation, cross-bridge cycling rate increases spontaneously, a phenomenon not previously reported. We speculate that the rapid re-elongation in late relaxation may reactivate muscle.

The effects of biofeedback-assisted relaxation on cell-mediated immunity, cortisol, and white blood cell count in healthy adult subjects.

The effect of biofeedback-assisted relaxation on cell-mediated immunity, cortisol, and white blood cell count was investigated in healthy adults under low-stress conditions. Fourteen subjects were trained with biofeedback-assisted relaxation for 4 weeks, while 17 subjects were controls. The group trained in relaxation techniques showed increased blastogenesis, decreased white blood cell count, due to decreased neutrophils, and no change in cortisol in comparison to the control group. Subjects with lower initial anxiety scores and forehead muscle tension levels showed larger increases in blastogenesis and larger decreases in neutrophils than subjects with higher initial anxiety and muscle tension levels.