Peter R. A. Johnson

Everolimus and mycophenolate mofetil are potent inhibitors of fibroblast proliferation after lung transplantation.

Background. Dysregulated fibroblast proliferation is thought to play an important role in the progression of bronchiolitis obliterans (BO) after lung transplantation. Augmented immunosuppression is often used to treat BO. We investigated the effect of methylprednisolone (mPRED), cyclosporine A (CsA), tacrolimus (FK506), azathioprine (AZA), mycophenolate mofetil (MMF), and everolimus (rapamycin derivative [RAD]) on the proliferative capacity of fibroblasts cultured from transbronchial biopsies of lung transplant recipients. Methods. Primary cultures of human lung fibroblasts were obtained from 14 transbronchial biopsies of lung transplant recipients. Subconfluent cells were serum starved for 24 hr followed by growth stimulation in the presence or absence of the respective drug in six concentrations ranging as follows: 0.01 to 100 mg/L for mPRED; 0.01 to 50 mg/L for CsA and AZA; 0.001 to 5 mg/L for FK506 and MMF; and 0.00001 to 1 mg/L for RAD. Proliferation was quantified by [3H]thymidine incorporation and direct cell count. A toxic drug effect was excluded by trypan blue. Results. Drug concentrations (mg/L) causing a 50% inhibition of fibroblast proliferation were mPRED 4; CsA 20; FK506 0.3; AZA 7; MMF 0.3; and RAD 0.0006. Drug concentrations (mg/L) causing inhibition of fetal bovine serum-induced proliferation were mPRED 60; CsA 45; FK506 3; AZA 35; MMF 1; and RAD 0.003. Conclusions. RAD and MMF were the most potent antifibroproliferative drugs and were effective at concentrations achieved clinically, supporting their use for the treatment of patients with early BO. Our method holds promise as an in vitro model to assess the likely in vivo responses of human lung fibroblasts to specific immunosuppressive drugs.

Mast cell tryptase potentiates histamine-induced contraction in human sensitized bronchus

The mast cell plays a pivotal role in the early asthmatic response via release of mediators, which directly influence airway smooth muscle tone. Canine mast cell tryptase has been reported to potentiate the contractile response of canine isolated airways to histamine. The aim of this study was to investigate whether human mast cell tryptase potentiated contractile responses in human isolated bronchi. The effect of tryptase differed according to the sensitization status of the bronchi. In lung tissue from sensitized patients (those whose bronchial tissue contracted in response to the application of any of four common antigens) 90 ng.mL-1 of human purified lung tryptase markedly potentiated the contractile response to histamine. The maximal response as a percentage of maximal contraction to acetylcholine was 80 +/- 8% in control tissues and 119 +/- 6% in tryptase treated tissues (n = 4; p < 0.05). Tryptase, at a dose of 200 ng.mL-1, also potentiated responses but to a lesser degree, 100 +/- 5% (n = 4; p < 0.05). In nonsensitized bronchi, neither 90 nor 200 ng.mL-1 tryptase had any significant effect on histamine responses. The increased response in the presence of tryptase in sensitized tissue was inhibited by the calcium voltage-dependent channel antagonist, verapamil (10(-6) M). We have shown, for the first time, that human mast cell tryptase potentiates contraction in sensitized bronchi via a calcium-related mechanism. These findings provide a link between a mast cell derived product and in vitro human airway hyperresponsiveness.

Sensitization alters contractile responses and calcium influx in human airway smooth muscle

Although an abnormality in airway smooth muscle has been promoted as a mechanism for airway hyperresponsiveness, there is, so far, little evidence to support this. We investigated whether in vitro hyperresponsiveness to pharmacologic agents could be induced in human airway tissue by passive sensitization and whether these changes in contractile responses were related to an alteration in calcium mobilization. Human bronchial tissue was incubated in serum with a high RAST titer to Dermatophagoides farinae. Control tissues were incubated in serum taken from a skin test-negative donor with a total IgE of less than 10 IU/ml. We compared contractile responses to histamine, KCI, and carbachol in nonsensitized and sensitized tissues and examined the effect on these responses of the calcium voltage-dependent channel agonist, BAY K8644 (10(-6) mol/L). We found that sensitization significantly increased responses to histamine, depressed responses to carbachol, and increased the involvement of the calcium voltage-dependent channel in contractions to KCl. These results suggest that airway hyperresponsiveness may be associated with altered calcium mobilization in airway smooth muscle.

Interleukin-4 inhibits mitogen-induced proliferation of human airway smooth muscle cells in culture

The increase in the amount of airway smooth muscle in the bronchial wall associated with asthma is partly due to hyperplasia. It is therefore important to determine which factors regulate growth and especially proliferation. In this study, we describe the effect of interleukin-4 (IL-4), a mast cell- and T lymphocyte-derived cytokine, on human airway smooth muscle proliferation as determined by [3H]thymidine uptake in the presence of fetal bovine serum (FBS), platelet-derived growth factor, basic fibroblast growth factor, and thrombin. IL-4 (5, 15, 50, and 150 ng/ml) significantly decreased 10% FBS-induced proliferation by 50, 73, 43, and 46%, respectively. The proliferative responses to platelet-derived growth factor (20 and 40 ng/ml), basic fibroblast growth factor (30 ng/ml), and thrombin (1 and 10 U/ml) were significantly reduced by 19, 21, 37, 36, and 57% respectively in the presence of 50 ng/ml of IL-4. We investigated the effect of IL-4 and other known inhibitors of smooth muscle proliferation, namely PGE2, heparin, and forskolin, on intracellular cAMP concentrations. IL-4 (50 ng/ml) and heparin (100 U/ml) did not alter intracellular cAMP levels when cells were treated with 1 or 10% FBS. PGE2 (1 μM) and forskolin (10 μM) significantly increased cAMP concentration above the control value in nonproliferating cells (1% FBS treated) by 7- and 37-fold, respectively. The effect of IL-4 (50 ng/ml), PGE2 (1 μM), and forskolin (10 μM) on cyclin D1 protein expression in 10% FBS-stimulated human airway smooth muscle cells was also examined. PGE2 and forskolin did not significantly inhibit cyclin D1 expression. However, IL-4 decreased cyclin D1 expression by 21%. These results provide evidence that IL-4 decreases human airway smooth muscle cell proliferation via a mechanism that is cAMP independent and mediated, in part, by a decrease in cyclin D1 protein expression.The increase in the amount of airway smooth muscle in the bronchial wall associated with asthma is partly due to hyperplasia. It is therefore important to determine which factors regulate growth and especially proliferation. In this study, we describe the effect of interleukin-4 (IL-4), a mast cell- and T lymphocyte-derived cytokine, on human airway smooth muscle proliferation as determined by [3H]thymidine uptake in the presence of fetal bovine serum (FBS), platelet-derived growth factor, basic fibroblast growth factor, and thrombin. IL-4 (5, 15, 50, and 150 ng/ml) significantly decreased 10% FBS-induced proliferation by 50, 73, 43, and 46%, respectively. The proliferative responses to platelet-derived growth factor (20 and 40 ng/ml), basic fibroblast growth factor (30 ng/ml), and thrombin (1 and 10 U/ml) were significantly reduced by 19, 21, 37, 36, and 57% respectively in the presence of 50 ng/ml of IL-4. We investigated the effect of IL-4 and other known inhibitors of smooth muscle proliferation, namely PGE2, heparin, and forskolin, on intracellular cAMP concentrations. IL-4 (50 ng/ml) and heparin (100 U/ml) did not alter intracellular cAMP levels when cells were treated with 1 or 10% FBS. PGE2 (1 microM) and forskolin (10 microM) significantly increased cAMP concentration above the control value in nonproliferating cells (1% FBS treated) by 7- and 37-fold, respectively. The effect of IL-4 (50 ng/ml), PGE2 (1 microM), and forskolin (10 microM) on cyclin D1 protein expression in 10% FBS-stimulated human airway smooth muscle cells was also examined. PGE2 and forskolin did not significantly inhibit cyclin D1 expression. However, IL-4 decreased cyclin D1 expression by 21%. These results provide evidence that IL-4 decreases human airway smooth muscle cell proliferation via a mechanism that is cAMP independent and mediated, in part, by a decrease in cyclin D1 protein expression.

Dual ERK and phosphatidylinositol 3-kinase pathways control airway smooth muscle proliferation: differences in asthma

Hyperplasia of airway smooth muscle (ASM) within the bronchial wall of asthmatic patients has been well documented and is likely due to increased muscle proliferation. We have shown that ASM cells obtained from asthmatic patients proliferate faster than those obtained from non‐asthmatic patients. In ASM from non‐asthmatics, mitogens act via dual signaling pathways (both ERK‐ and PI 3‐kinase‐dependent) to control growth. In this study we are the first to examine whether dual pathways control the enhanced proliferation of ASM from asthmatics. When cells were incubated with 0.1% or 1% FBS, ERK activation was significantly greater in cells from asthmatic subjects (P < 0.05). In contrast, when cells were stimulated with 10% FBS, ERK activity was significantly greater in the non‐asthmatic cells. However, cell proliferation in asthmatic cells was still significantly higher in cells stimulated by both 1% and 10% FBS. Pharmacological inhibition revealed that although dual proliferative pathways control ASM growth in cells from non‐asthmatics stimulated with 10% FBS to an equal extent ([3H]‐thymidine incorporation reduced to 57.2 ± 6.9% by the PI 3‐kinase inhibitor LY294002 and 57.8 ± 1.1% by the ERK‐pathway inhibitor U0126); in asthmatics, the presence of a strong proliferative stimulus (10% FBS) reduces ERK activation resulting in a shift to the PI 3‐kinase pathway. The underlying mechanism appears to be upregulation of an endogenous MAPK inhibitor—MKP‐1—that constrains ERK signaling in asthmatic cells under strong mitogenic stimulation. This study suggests that the PI 3‐kinase pathway may be an attractive target for reversing hyperplasia in asthma. J. Cell. Physiol. 216: 673–679, 2008,

Characterization of contractile prostanoid receptors on human airway smooth muscle

In human bronchial rings the thromboxane A2 (TxA2) mimetic, U46619, produced cumulative concentration-related contractions up to a maximum of 141 +/- 23% of the response induced by carbachol or acetylcholine. The geometric mean EC50 value was 3.2 X 10(-8) M (95% confidence interval: 1.2, 8.9 X 10(-8) M) (n = 5). Contractions to U46619 were unaffected by atropine (10(-6) M) or verapamil (10(-5) M), but were competitively antagonized by the TxA2 antagonist GR32191 with a pA2 value of 8.40 +/- 0.41. The maximum contractile response to prostaglandin (PG) F2 alpha was smaller (90 +/- 9%, n = 13) and the potency was less (EC50 = 2 X 10(-6) M) than that of U46619. Contractions to PGF2 alpha were also competitively antagonized by GR32191 with a pA2 value of 8.18 +/- 0.08. Concentration-response curves to PGE2 were biphasic, relaxation at concentrations from 10(-9) to 10(-6) M and contraction from 10(-6) to 3 X 10(-5) M. GR32191 10(-7) M inhibited the contractile portion of the response curve in 8 of 11 tissues. Based on these results we conclude that U46619, PGF2 alpha and PGE2 all contract human airways by stimulation of the TxA2 (TP) receptor.

Airway smooth muscle--its relationship to the extracellular matrix.

The airway smooth muscle cell has a variety of properties, which confer on it the ability to participate actively in the inflammatory process and the remodeling events, which accompany severe, persistent asthma. Among these properties is its relationship to the extracellular matrix (ECM) with which it interacts by releasing matrix proteins, matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs). Muscle cells derived from asthmatic subjects proliferate faster, release a different profile of matrix proteins, produce more connective tissue growth factor (CTGF) in response to TGFbeta stimulation and these changes may impact on airway smooth muscle contraction and proliferation. Integrins on the surface of the airway smooth muscle transduce signals between the muscle cell and the ECM, but whether the expression and/or function of these is altered in asthma is not known. It is unlikely that current therapy is effective in preventing or reversing remodeling, and therefore, understanding the pathophysiological events, which underlie its mechanism is critical.

Potentiation of the contractile effects of neuropeptides in human bronchus by an enkephalinase inhibitor

Many neuropeptides have been identified in human airways and these are susceptible to breakdown by endogenous enkephalinases. This study investigated the effect of enkephalinase inhibition with phosphoramidon (10(-5) M) on contractile responses to neurokinin A, eledoisin, physalaemin and substance P in human isolated bronchial smooth muscle. Contractile responses to the maximal doses of neuropeptides given were potentiated by phosphoramidon, whereas those to carbachol were unaffected. In addition, neuropeptide response curves were significantly (P less than 0.05) shifted to the left as measured by geometric mean dose ratios (95% confidence intervals) of 20.9 (5.4-81.3) n = 7 for neurokinin A; 63 (21.8-181.9) n = 6 for eledoisin, 44.7 (3.2-616.6) n = 5 for physalaemin and 6.9 (2.4-20) n = 6 for substance P. We conclude that enkephalinase inhibition in vitro significantly potentiates the contractile response to neuropeptides in human airway smooth muscle. Absence of, or decreased activity of enkephalinase could lead to enhanced effects of endogenous neuropeptides in human airways in vivo.

Tumour necrosis factor-α potentiates contraction of human bronchus in vitro

Chronic inflammation of the airways is an important component in the induction of airway hyperresponsiveness (AHR) in asthma. The pro‐inflammatory cytokines interleukin‐1β (IL‐1β) and tumour necrosis factor‐α (TNF‐α) have been implicated in the induction of AHR. Whether these cytokines directly modulate the contractile properties of human airway smooth muscle (ASM) has not been fully investigated.

Human eosinophil–airway smooth muscle cell interactions

Eosinophils are present throughout the airway wall of asthmatics. The nature of the interaction between human airway smooth muscle cells (ASMC) and eosinophils was investigated in this study. We demonstrated, using light microscopy, that freshly isolated eosinophils from healthy donors rapidly attach to ASMC in vitro. Numbers of attached eosinophils were highest at 2 h, falling to 50% of maximum by 20 h. Eosinophil attachment at 2 h was reduced to 72% of control by anti-VCAM-1, and to 74% at 20 h by anti-ICAM-1. Pre-treatment of ASMC for 24h with TNF-alpha, 10 nM, significantly increased eosinophil adhesion to 149 and 157% of control after 2 and 20 h. These results provide evidence that eosinophil interactions with ASMC involve VCAM-1 and ICAM-1 and are modulated by TNF-alpha.