The Journal of Allergy and Clinical Immunology
Volume 104, Issue 2 , Pages 294-300, August 1999

Proteasome inhibition: A novel mechanism to combat asthma☆☆☆

Cambridge, Mass, and Edinburgh, United Kingdom

From aProScript, Inc, Cambridge; and bQuintiles Ltd, Edinburgh

Received 2 February 1999; received in revised form 31 March 1999; accepted 31 March 1999.

Article Outline

Abstract 

Background: Nuclear factor-κB (NF-κB) is a critical transcription factor required for the regulation of many genes involved in inflammatory responses to noxious stimuli. On activation, NF-κB induces the transcription of numerous proinflammatory cytokines, enzymes, and cellular adhesion molecules. Blockade of the proteasome with selective inhibitors attenuates the effects of NF-κB, leading to suppression of the inflammatory response. Objective: We sought to determine whether proteasome inhibitors would be active in a model of asthma. Methods: The mouse delayed-type hypersensitivity model was used to screen a panel of compounds for in vivo activity. The proteasome inhibitor, PS-519, was shown to be the most active in this model and was selected for further development. Allergen-induced pulmonary eosinophilia in Brown Norway rats was used subsequently to determine anti-inflammatory activity in an animal model. Results: Direct administration of PS-519 into the lungs significantly reduced leukocyte numbers, particularly the selective increase in eosinophils. Because steroids are the mainstay anti-inflammatory therapy in asthma, and data is available to suggest their possible interaction to suppress the activation of NF-κB, rats were also treated by inhalation with combinations of a steroid and the proteasome inhibitor. In both the delayed-type hypersensitivity and the animal eosinophil model, low doses of proteasome inhibitors were shown to be effective when given with low doses of steroids. Conclusion: Taken together, the present data suggest that proteasome inhibition may represent a novel strategy for the treatment of inflammatory lung diseases such as asthma. (J Allergy Clin Immunol 1999;104:294-300.)

Keywords:  Asthma, cell adhesion molecules, cytokines, delayed-type hypersensitivity, eosinophils, leukocytes, nuclear transcription factor-κB, proteasome, steroids

Abbreviations:  DNFB , 2,4-Dinitrofluorobenzene, DTH , Delayed-type hypersensitivity, IκB , Inhibitor factor-κB, NF-κB , Nuclear transcription factor-κB

 

Asthma is a chronic inflammatory condition of the airways, which are hyperreactive and constrict easily in response to diverse stimuli, giving rise to reversible airways obstruction.1 This disease affects 5% to 10% of the population in industrialized countries, and there is good evidence that the prevalence and severity of asthma are rising.2 The disease is characterized by a significant inflammatory cell infiltrate into the airways, which is comprised predominantly by eosinophils and TH2 cells.3, 4 Furthermore, in asthmatic subjects there is clear evidence of plasma exudation, airway mucosal edema, smooth muscle hyperplasia and hypertrophy, mucous plugging, and epithelial damage.1, 3, 5, 6, 7 These inflammatory events are orchestrated by activated infiltrating eosinophilic granulocytes and other infiltrating cells, such as lymphocytes, through the release of multiple inflammatory mediators, including cytokines that further propagate the disease.1, 4

Initially, primed eosinophils interact with cell adhesion molecules present on endothelial cells lining the vasculature to allow them to undergo diapedesis into the airways.8 Once activated, eosinophils in the airway mucosa can generate a range of inflammatory mediators, including superoxide, histamine, leukotrienes, and platelet-activating factor.9, 10, 11, 12, 13, 14, 15 Moreover, eosinophils have the capacity to generate additional cytokines that amplify the response to allergen.16, 17, 18 Indeed, basic proteins released by activated eosinophils in the lung are lytic for airway epithelium19 and are considered to contribute to the characteristic lung pathology observed in asthma.1, 4 Hence several developmental strategies to reduce the recruitment, longevity, and activation of these cells are presently under investigation, including the use of animal models that exhibit clear eosinophilia.20, 21, 22

The synthesis of many of the mediators that orchestrate the inflammatory response in asthma are under the control of nuclear transcription factor-κB (NF-κB).23 Levels of this intracellular factor are under the control of inhibitor factor-κB (IκB), which forms a complex with NF-κB and masks the nuclear translocation sequence on NF-κB.24, 25, 26, 27 IκB masks the nuclear translocation sequence on NF-κB, thereby blocking its passage into the nucleus. After stimulation of the cell (eg, by environmental stress or cytokines), IκB becomes phosphorylated and then bound to polyubiquitin chains.28, 29, 30 These ubiquitin chains identify the protein IκB as a substrate for degradation by the proteasome, which exists in all eukaryotic cells.31, 32, 33 After the degradation of IκB, NF-κB is subsequently released34, 35 and is free to pass into the nucleus where it can elicit the transcription of multiple genes, leading to the synthesis of many proinflammatory proteins.34, 35 As such, NF-κB regulates the expression of multiple cytokines, as well as cellular adhesion molecules, responsible for the migration of cells from the circulation into the airways. Hence NF-κB amplifies the inflammation and is critically involved in the recruitment of a number of leukocytes, including T helper cells and eosinophils, into the lungs.

In addition to controlling cytokine and cell adhesion molecule expression, nitric oxide synthetase is also under the transcriptional regulation of NF-κB. Nitric oxide is released from airway epithelium, where it plays an important regulatory role in normal and pathophysiologic airway function.36 Elevated levels of nitric oxide synthetase are found in epithelial cells of asthmatic subjects stimulated with cytokines, and evidence of increased nitric oxide production in such patients has also been reported.36 Together, these data add further support for the concept of turning down NF-κB activity by means of proteasome inhibition as a mechanism to reduce the inflammatory effects of this factor.

Currently, steroids are the mainstay anti-inflammatory therapy in asthma.37, 38, 39, 40 Such agents exert their therapeutic effects by interacting with cellular glucocorticoid receptors to modify the inflammatory response.41 Recent evidence has suggested that steroids may also interact with the ubiquitin-proteasome pathway, and in particular NF-κB activity, and therefore could act synergistically with proteasome inhibitors to enhance their anti-inflammatory activity.

Although lactacystin was discovered in 1991 by Omura et al,42 it was not until 1995 that its activity as a proteasome inhibitor was elucidated.43 Subsequently, the mechanism by which lactacystin enters cells and inhibits the enzyme, as a β-lactone derivative, was reported.44, 45 Because of the inability of lactacystin to penetrate cells and its short half-life in aqueous solution, an effort to synthesize clasto-lactacystin-β-lactone (β-lactone) derivatives has been underway. Recently, a new class of selective and potent inhibitors of the proteasome were synthesized with a goal of exploring their potential anti-inflammatory effects.46 These compounds were designed to attenuate the proteolytic activity of the proteasome and inhibit the degradation of IκB, thereby reducing the effects of NF-κB. As a consequence, such compounds are expected to decrease cytokine release and inhibit stimulated cellular adhesion molecule expression.47

The current investigation reports on the effects of a number of proteasome inhibitors in a primary screen used to determine in vivo activity (ie, the delayed-type hypersensitivity [DTH] model). Subsequently, one such compound, PS-519, was examined in allergen-induced pulmonary eosinophilia in the actively sensitized Brown Norway rat, in which interactions between the inhibitor and the steroid, budesonide, were explored. This model was chosen because previous reports have established that actively sensitized, allergen-challenged rats exhibit significant lung eosinophilia20, 21, 22 that is characteristic of the lungs of asthmatic subjects.1, 4

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METHODS 

Animals 

Male BALB/c mice (18 to 20 g) were purchased from Charles River (Wilmington, Mass) and housed in groups of 5 per cage. Pellets of standard rodent chow (#5001; Purina, St Louis, Mo) and tap water were available ad libitum throughout the studies. All procedures were approved by the Institutional Animal Use and Care Committee in accordance with National Institutes of Health Guidelines.

Male Brown Norway rats (180 to 200 g) were supplied by Harlan Olac Limited (Bicester, UK). Animals were housed in groups of 5 per cage and provided with a diet of RMI (E) SQC (Special Diets Services, Witham, UK) and tap water. Both were freely available during the studies.

Drugs 

Proteasome inhibitors (including PS-519) were synthesized in the Chemistry Department at ProScript, Inc (Cambridge, Mass). Other agents were purchased from Sigma (St Louis, Mo) unless otherwise stated.

PS-519 was initially made up as a 2× stock in 100% propylene glycol and then diluted 1:1 with 0.9% saline immediately before administration. Dexamethasone was made up in 0.9% saline. The appropriate vehicle was used as a control for each test agent.

Fluorometric 20S proteasome assay 

Test drugs were added to 2 mL of substrate buffer (20 mmol/L HEPES, 0.5 mmol/L EDTA, 0.035% SDS, and 60 μmol/L Ys substrate; pH 8.0, 37°C), and the rate of substrate cleavage/20S proteasome activity was determined. Succinyl-Leu-Leu-Val-Tyr-Amido-4-methyl-coumarin (Ys substrate) was obtained from Bachem (King of Prussia, Pa). This assay has been previously reported for the evaluation of compounds for proteasome inhibition.48

Delayed-type hypersensitivity 

This procedure has been previously described elsewhere.49 Briefly, 2,4-dinitrofluorobenzene (DNFB) was dissolved in 4:1 acetone/olive oil vehicle and applied topically (20 μL/foot; 0.5%) to both hind limb footpads of mice on days 0 and 1. A pipette tip was used to distribute the solution evenly on the feet, which were then dried with warm air before returning each animal to its cage. On day 5, the mice were anesthetized with ketamine/xylazine (80/16 mg/kg, respectively) and then challenged with DNFB (0.2%), which was applied topically to the inner and outer surfaces of the left ear (10 μL each). The vehicle was applied to the right ear of all mice.

For drug studies, the mice were administered the test agent intravenously (2.5 mL/kg) 30 minutes before DNFB challenge on day 5. On day 6, the mice were reanesthetized, and the thickness of both ears was measured with calipers. The difference between the drug (left) and the vehicle-treated (right) ears was then recorded.

Allergen-induced pulmonary eosinophilia model 

After acclimatization for at least 5 days, male Brown Norway rats (180 to 200 g) were injected (0.5 mL administered intraperitoneally) with ovalbumin (10 μg) mixed with aluminium hydroxide gel (10 mg), and this procedure was repeated 7 and 14 days later. On day 21, rats were anaesthetized (halothane 5% in O2 ), and either vehicle, PS-519, budesonide, or a combination of budesonide and PS-519 (approximately 1.0 mg of powder/animal) was instilled through a cannula connected to a Penn-Century delivery device (PennCentury, Philadelphia, Pa) placed directly into the trachea 1 hour before ovalbumin exposure; this procedure was repeated 24 and 48 hours after ovalbumin exposure. After recovery, sensitized animals were restrained in plastic tubes and exposed (60 minutes) to an aerosol of ovalbumin (10 mg/mL) in a nose-only exposure system.50 Animals were killed 72 hours later with pentobarbital (250 mg/kg administered intraperitoneally). The lungs underwent lavage with 3 aliquots (4 mL) of HBSS (HBSS × 10, 100 mL; EDTA 100 mmol/L, 100 mL; HEPES 1 mol/L, 10 mL made up to 1.0 L with water); recovered cells were pooled, and the total volume of recovered fluid was adjusted to 12 mL by addition of HBSS. Total cells were counted (Sysmex Microcell Counter F-500, TOA Medical Electronics Ltd). Smears were made by diluting recovered fluid (to approximately 106 cells/mL) and spinning an aliquot (100 μL) in a centrifuge (Cytospin, Shandon, UK). Smears were air-dried, fixed with a solution of fast green in methanol (2 mg/L) for 5 seconds, and stained with eosin G (5 seconds) and thiazine (5 seconds; Diff-Quik, Baxter Dade Ltd, Switzerland) to differentiate eosinophils, neutrophils, macrophages, and lymphocytes. A total of 500 cells per smear were counted by light microscopy under oil immersion (×1000).

Statistical analysis 

Data were analyzed by using an ANOVA followed by a post-hoc Dunnett’s t test. By using 2-tailed tables, P values of less than .05 were considered significant.

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RESULTS 

In vitro testing of β-lactone and its synthetic analogs 

The rank order of various β-lactone analogs was prepared from data demonstrating the ability of each compound to inhibit the proteasome-induced hydrolysis of a peptide substrate in vitro by using a previously reported assay system.48 The results are presented in Table I and show that PS-519 was the most active compound in the series.

Table I. Structure-activity profile of several proteasome inhibitors based on β-lactone
CompoundSubstitutionActivity
β-LactoneMe20,000
PS-399H4500
PS-401OH8900
PS-409Et39,000
PS-519n-Pr46,500
PS-536n-Bu38,000
PS-539Ch2 Ph6400

The substitution is for the alkyl group in (1R-[1S,4R,5S])-1-(1-hydroxydroxy-2-methylpropyl)-4-alkyl-6-oxa-2-azabicyclo(3.2.0)heptane-3,7-dione. The activity values represent kobs /(inhibitor concentration) in mol L–1 s-1 for the results from the in vitro proteasome assay.

As such, PS-519 was further explored in our in vivo models.

Effects in the DTH model 

Mice were sensitized with DNFB as described above and then treated with an intravenous bolus of test agent for 30 minutes before the final challenge. Ear swelling was recorded at 24 hours by using calipers and compared with contralateral (control) ear measurements. The difference between the 2 ears was recorded, and values from each drug-treated mouse were compared with those from vehicle-treated animals. Data show that dexamethasone (0.03 to 1.0 mg/kg; n = 8 to 18/group) elicited a clear dose-dependent decrease in ear swelling, which reached significance (P < .01) at 0.3 mg/kg (Fig 1).

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  • Fig. 1. 

    Effect of intravenous administration of dexamethasone on ear swelling in mice induced by treatment with DNFB. Dexamethasone was given 30 minutes before challenge. Values are given as percent of vehicle (Veh) -treated mice ± SEM. *P < .01 with respect to vehicle-treated animals (n = 8 to 18/group).

Identical studies were then repeated by using the proteasome inhibitors, β-lactone (0.01 to 10 mg/kg; n = 5 to 201/group), or PS-519 (0.03 to 1.0 mg/kg; n = 26 to 131/group). Again, results showed that both inhibitors elicited dose-dependent decreases in ear swelling compared with vehicle-treated mice. The dose at which statistically significant (P < .01) effects were seen after treatment with either β-lactone or PS-519 was 0.1 mg/kg (Figs 2 and 3).
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  • Fig. 2. 

    Effect of intravenous administration of the proteasome inhibitor β-lactone on ear swelling in mice induced by treatment with DNFB. β-Lactone was given 30 minutes before challenge. Values are given as percent of vehicle (Veh) -treated mice ± SEM. *P < .01 with respect to vehicle-treated animals (n = 5 to 201/group).

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  • Fig. 3. 

    Effect of intravenous administration of the proteasome inhibitor PS-519 on ear swelling in mice induced by treatment with DNFB. PS-519 was given 30 minutes before challenge. Values are given as percent of vehicle (Veh) -treated mice ± SEM. *P < .01 with respect to vehicle-treated animals (n = 26 to 131/group).

Of great interest was the finding that a decrease in ear swelling (P < .01) was also obtained with low doses of β-lactone (0.01 or 0.03 mg/kg) when combined with a no-effect dose (0.3 mg/kg) of dexamethasone (Fig 4).
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  • Fig. 4. 

    Effect of intravenous administration of the proteasome inhibitor β-lactone (Lact) in combination with dexamethasone (Dex) on ear swelling in mice induced by treatment with DNFB. Both drugs were given 30 minutes before challenge. Values are given as percent of vehicle (Veh)-treated mice ± SEM. *P < .05 with respect to vehicle-treated animals; #P < .05 with respect to dexamethasone-treated animals (n = 16 /group).

These data clearly support the use of such drugs in combination to maximize the therapeutic window (ie, maintaining efficacy with a decrease in side-effect potential).

Effects on allergen-induced pulmonary eosinophilia 

Exposure of actively sensitized Brown Norway rats to ovalbumin induced a significant increase in the total number of leukocytes recovered in lung lavage fluid (Fig 5), which was mainly the result of an increase in eosinophil numbers (Fig 6).

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  • Fig. 5. 

    Effect of intratracheal administration of the proteasome inhibitor PS-519 on infiltration of leukocytes into the lungs of Brown Norway rats challenged with ovalbumin. PS-519 was given 1 hour before and 24 and 48 hours after challenge. Cell number values are given as means ± SEM. *P < .05 and **P < .01 with respect to vehicle (Veh)-treated animals (n = 10/treatment group and n = 5 for the naive group).

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  • Fig. 6. 

    Effect of intratracheal administration of the proteasome inhibitor PS-519 on eosinophilia in lungs of Brown Norway rats challenged with ovalbumin. PS-519 was given 1 hour before and 24 and 48 hours after challenge. Cell number values are given as means ± SEM. *P < .05 and **P < .01 with respect to vehicle (Veh)-treated animals (n = 10/treatment group and n = 5 for the naive group).

Intratracheal administration of PS-519 (0.03 to 0.3 mg/kg; n = 10/group) before and after ovalbumin exposure produced a clear, dose-related, and significant (P < .05) reduction not only in the total number of leukocytes in the sensitized lungs (Fig 5) but importantly in the number of eosinophils (Fig 6). Modest effects were seen at a dose of 0.1 mg/kg, whereas more robust reductions in cell infiltrate were observed at a dose of 0.3 mg/kg. Of importance, when sensitized rats were treated with a combination of PS-519 (0.03 or 0.1 mg/kg; n = 8/group) and the glucocorticoid budesonide (0.1 mg/kg), there was also a clearly significant (P < .05) attenuation of the eosinophilia (Fig 7), even though the doses of either compound, when administered alone, had modest or no activity.
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  • Fig. 7. 

    Effect of intratracheal administration of the proteasome inhibitor PS-519 on eosinophilia in lungs of Brown Norway rats challenged with ovalbumin in combination with the corticosteroid budesonide (0.1 mg/kg). PS-519 was given 1 hour before and 24 and 48 hours after challenge. Cell number values are given as means ± SEM. *P < .05 with respect to vehicle-treated animals; #P < .05 with respect to budesonide-treated rats (n = 5 to 8/group).

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DISCUSSION 

The results from this study demonstrate a novel mechanism, proteasome inhibition, which may have potential therapeutic benefit for asthmatic subjects. The data clearly show that the proteasome inhibitor, PS-519, significantly reduced the infiltration of eosinophils into the antigen-challenged lung, where they are known to contribute to the inflammatory process.1, 4 Furthermore, when PS-519 was given in combination with a no-effect dose of the steroid budesonide, enhanced anti-inflammatory activity was observed. These data therefore not only support the potential use of PS-519 as a single agent anti-inflammatory therapy but also highlight the possibility of combination treatment regimens, where it could be given with currently used steroids such as budesonide.

Many inflammatory conditions involve a self-perpetuating cascade reaction involving thymus-derived lymphocytes, cytokines, and a variety of soluble inflammatory mediators.51 In some circumstances, specific stimuli elicit an inflammatory response, which becomes exacerbated with a subsequent stimulus challenge. This series of events is often referred to as DTH or contact sensitivity.52, 53, 54 Therefore we assessed the anti-inflammatory activity of agents in the DTH model as a preliminary step for their testing in more stringent chronic inflammatory models as part of their path toward clinical development for major chronic diseases such as asthma. That β-lactone and PS-519 were effective in the DTH model warranted further testing in more demanding and potentially appropriate clinical models.

Furthermore, it is known that various cell adhesion molecules are also under the regulation by NF-κB, and as such, their expression is also decreased by proteasome inhibition.55 As a consequence, inflammatory cell infiltration would be limited and the overall inflammatory response reduced.56, 57, 58 This entire cascade of events can be attenuated and even blocked by reducing the enzymatic activity of the proteasome with specific inhibitors. As such, a reduction of NF-κB activity would lead to a decreased cytokine expression and release and an overall attenuation of the inflammatory response. Indeed, another proteasome inhibitor, PS-341, was recently shown to be very effective at reducing not only the development of the disease but also a number of the inflammatory mediators in an inflammatory model of arthritis.59 The present results extend these findings such that the proteasome inhibitor, PS-519, not only reduced the ear swelling in the DTH model but also significantly reduced the cellular infiltration in the lungs of sensitized rats after allergen exposure.

Steroids are currently the mainstay anti-inflammatory therapy in the treatment of asthma. However, it is well known that prolonged systemic administration of these drugs has been associated with a number of adverse side effects, including osteoporosis, suppression of the hypothalamic-pituitary-adrenal axis, cataracts, skin thinning, suppression of growth, and candidiasis.39 Although low inhaled doses of steroids have less adverse effects, a similar profile to systemic drug administration can occur with higher inhaled doses.60, 61 There is now a growing body of evidence that supports the potential for synergistic activity between glucocorticoids and proteasome inhibitors. As such, it is known that activated glucocorticoid receptors not only lead to a suppression of transcription of inflammatory cytokines62, 63 and increase the instability of transcript products64 but also increase intracellular levels of IκBα.65, 66 Steroids, such as dexamethasone and budesonide, would be expected to reduce NF-κB activity by increasing intracellular levels of the inhibitory protein IκB. Moreover, activation of glucocorticoid receptors have been reported to physically inhibit NF-κB binding to its recognition site on DNA.67, 68, 69, 70 Together, these steroid-induced effects would limit the transcriptional activity of NF-κB and thereby reduce the inflammatory response after its activation (Fig 8).

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  • Fig. 8. 

    Mechanism by which proteasome inhibitors and corticosteroids could interact in a cell to elicit their anti-inflammatory activity. The illustration shows that, once activated, IκB is phosphorylated and then ubiquitinated before its degradation by the proteasome. This process then releases NF-κB, which translocates to the nucleus and binds to sites that initiate the transcription of IκB along with the induction of many proinflammatory molecules. This new IκB can then move into the cytoplasm to sequester free NF-κB, thereby resetting the inflammatory switch. PS-519 blocks the degradation of IκB at the level of the proteasome and hence inhibits activation of NF-κB and elicits its anti-inflammatory action. In addition, corticosteroids, such as budesonide, can bind to cytoplasmic steroid receptors, which move to the nucleus and bind to glucocorticosteroid response elements (GRE) . Activation of GRE leads to the transcription of IκB.

Proteasome inhibitors, such as PS-519, would also decrease NF-κB activity by attenuating the degradation of the inhibitory protein IκB. Hence it is conceivable that treatment schedules with low doses of each compound would have significant anti-inflammatory effects with limited side effects. The present studies set out to examine the effects of selective and potent proteasome inhibitors in the presence and absence of steroids in the DTH reaction and an animal model of allergen-induced eosinophilia. Results from the current studies clearly show additive or even synergistic activity in both models. In the DTH model nonactive doses of dexamethasone and β-lactone were effective when given in combination to an extent equivalent to much higher doses of each compound given alone. More importantly, combination of low doses of budesonide and PS-519 were very effective at reducing the eosinophilia in the allergen-induced model. Finally, if lower doses of steroids can be used, with maintained efficacy, then their side-effect profile can be modified and could lead to a significant improvement in the quality of life of asthmatic patients.

Many clinical trials are currently evaluating therapies designed to block the activity of a single cytokine or cell adhesion molecule.71 Treatment regimens using proteasome inhibitors would offer the advantage of affecting multiple targets at once because many of these targets are under the transcriptional regulation of NF-κB.23 Taken together, these data demonstrate that PS-519 has significant therapeutic potential either as a single agent or in combination with current therapies to treat disease states such as asthma.

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References 

  1. Beasley R, Roche WR, Roberts JA, Holgate ST. Cellular events in the bronchi in mild asthma and after bronchial provocation. Am Rev Respir Dis. 1989;139:806–817
  2. Fleming BN, Crombie BL. Prevalence of asthma and hay fever in England and Wales. BMJ. 1987;294:279–283
  3. Djukanovic R, Roche WR, Wilson JW, Beasley CR, Twentyman OP, Howarth RH, et al.  Mucosal inflammation in asthma. Am Rev Respir Dis. 1990;142:434–457
  4. Busse WW. Inflammation in asthma: the cornerstone of the disease and target of therapy. J Allergy Clin Immunol. 1998;102:S17–S22
  5. Bousquet J, Chanez P, Lacoste LY, Barneon G, Ghavanian N, Enander I, et al.  Eosinophilic inflammation in asthma. N Engl J Med. 1990;323:1033–1039
  6. Robinson DS, Hamid Q, Ying S, Tsicopoulos A, Barkans J, Bentley AM, et al.  Predominant TH2-like bronchoalveolar T-lymphocyte population in atopic asthma. N Engl J Med. 1992;362:298–304
  7. Woolley KL, Adelroth E, Woolley MJ, Ellis R, Jordana M, O’Byrne PM. Granulocyte-macrophage colony-stimulating factor, eosinophils and eosinophil cationic protein in subjects with and without mild, stable, atopic asthma. Eur Respir J. 1994;7:1576–1584
  8. Montefort S, Holgate ST, Howarth PH. Leucocyte-endothelial adhesion molecules and their role in bronchial asthma and allergic rhinitis. Eur Respir J. 1993;6:1044–1054
  9. Owen WF, Rothenberg ME, Silberstein DS, Gasson JC, Stevens RL, Austen KF, et al.  Regulation of human eosinophil viability, density, and function by granulocyte/macrophage colony-stimulating factor in the presence of 3T3 fibroblasts. J Exp Med. 1987;166:129–141
  10. Rothenberg ME, Owen WF, Silberstein DS, Woods J, Soberman RJ, Austen KF, et al.  Human eosinophils have prolonged survival, enhanced functional properties, and become hypodense when exposed to interleukin 3. J Clin Invest. 1988;81:1986–1992
  11. Smith LJ. The role of platelet-activating factor. Am Rev Respir Dis. 1991;143:S100–S102
  12. Page CP. Mechanisms of hyperresponsiveness: platelet-activating factor. Am Rev Respir Dis. 1992;145:S31–S33
  13. Drazen JM, Gaston B, Shore S. Chemical regulation of pulmonary airway tone. Ann Rev Physiol. 1995;57:151–170
  14. Busse WW. The role and contribution of leukotrienes in asthma. Ann Allergy Asthma Immunol. 1998;81:17–26
  15. Kita H, Ohnishi T, Okubo Y, Weiler D, Abrams JS, Gleich GJ. Granulocyte/macrophage colony-stimulating and interleukin 3 release from human peripheral blood eosinophils and neutrophils. Exp Med. 1991;174:745–748
  16. Moqbel R, Hamid Q, Ying S, Barkans J, Hartnell A, Tsicopoulos A, et al.  Expression of mRNA and immunoreactivity for the granulocyte/macrophage colony-stimulating factor in activated human eosinophils. J Exp Med. 1991;174:749–752
  17. Drazen JM, Arm JP, Austen KF. Sorting out the cytokines of asthma. J Exp Med. 1996;183:1–5
  18. Drazen JM, Israel E, O’Byrne PM. Treatment of asthma with drugs modifying the leukotriene pathway. N Engl J Med. 1999;340:197–206
  19. Elwood W, Lotball JO, Barnes PJ, Fan-Chung K. The effect of dexamethasone and cyclosporine A on allergen-induced airway hyperresponsiveness and inflammatory cell responses in sensitized brown Norway rats. Am Rev Respir Dis. 1992;145:1289–1294
  20. Chapman ID, Lee AJ, Thompson D, Templeton AGB, Milne AAY. Time course of allergen-induced leucocyte accumulation in actively sensitized Brown Norway rats [abstract]. Am J Respir Crit Care Med. 1996;153:A219
  21. Chapman ID, Lee AJ, Thompson D, Gilmour L, Trotter L, Milne AAY. Allergen-induced airway hyperreactivity and eosinophil accumulation are temporally dissociated in actively sensitized brown Norway rats [abstract]. Am J Respir Crit Care Med. 1997;155:A881
  22. Frigas E, Loegering DA, Gleich DJ. Cytotoxic effects of the guinea pig eosinophil major basic protein on tracheal epithelium. Lab Invest. 1980;42:35–43
  23. Barnes PJ, Karin M. Nuclear factor-κB: a pivotal transcription factor in chronic inflammatory diseases. N Engl J Med. 1997;336:1066–1071
  24. Baeuerle PA, Baltimore D. IκB: a specific inhibitor of the NF-κB transcription factor. Science. 1988;242:540–546
  25. Matthews JR, Hay RT. Regulation of the DNA binding activity of NF-κB. Int J Cell Biol. 1995;27:865–879
  26. Thanos D, Maniatis T. NF-kappa B: a lesson in family values. Cell. 1995;80:529–532
  27. Baldwin AS. The NF-κB and IκB proteins: new discoveries and insights. Ann Rev Immunol. 1996;16(14):649–683
  28. Schreck R, Albermann K, Baeuerle PA. Nuclear factor κB: an oxidative stress-responsive transcription factor of eukaryotic cells. Free Rad Res Commun. 1992;17:221–237
  29. Chau V, Tobias JW, Bachmair A, Marriott D, Ecker DJ, Gonda DK, et al.  A multiubiquitin chain is confined to specific lysine in a targeted short-lived protein. Science. 1989;243:1576–1583
  30. Grilli MJ, Chiu J-S, Lenardo MJ. NF-κB and rel-participants in a multiform transcriptional regulatory system. Int Rev Cytol. 1993;143:1–63
  31. Goldberg AL, Stein R, Adams J. New insights into proteasome function: from archaebacteria to drug development. Chem Biol. 1995;2:503–508
  32. Coux O, Tanaka K, Goldberg AL. Structure and function of the 20S and 26S proteasomes. Ann Rev Biochem. 1996;65:801–847
  33. King RW, Deshaies RJ, Peters J-M, Kirschner MW. How proteolysis drives the cell cycle. Science. 1996;274:1652–1659
  34. Palombella VJ, Rando OJ, Goldberg AL, Maniatis T. The ubiquitin-proteasome pathway is required for processing the NF-kB1 precursor protein and the activation of NF-kB. Cell. 1994;78:773–785
  35. Chen Z, Hagler J, Palombella VJ, Melandri F, Scherer D, Ballard D, et al.  Signal-induced site-specific phosphorylation targets IB to the ubiquitin-proteasome pathway. Genes Dev. 1995;9:1586–1597
  36. Barnes PJ. Nitric oxide and airway disease. Ann Med. 1995;27:389–393
  37. Holgate ST, Djukanovic R, Wilson J, Roche W, Britten K, Howarth PH. Allergic inflammation and its pharmacological modulation in asthma. Int Arch Allergy Appl Immunol. 1991;94:210–217
  38. Barnes PJ. Molecular mechanisms of anti-asthma therapy. Ann Med. 1995;27:531–535
  39. Schimmer BP, Parker KL. Adrenocorticotropic hormone; adrenocortical steroids and their synthetic analogs; inhibitors of the synthesis and actions of adrenocortical hormones. In: 9th ed.  Hardman JG,  Limbird LE,  Malinoff PB,  Ruddon RW,  Gilman AG editor. Goodman and Gilman’s the pharmacological basis of therapeutics. 1995;p. 1459–1485
  40. Serafin WE. Drugs used in the treatment of asthma. In: 9th ed.  Hardman JG,  Limbird LE,  Malinoff PB,  Ruddon RW,  Gilman AG editor. Goodman and Gilman’s the pharmacological basis of therapeutics. 1995;p. 659–682
  41. Saatcioglu F, Claret F-X, Karin M. Negative transcriptional regulation by nuclear receptors. Semin Cancer Biol. 1994;5:347–359
  42. Omura S, Matsuzaki K, Fujimoto T, Kosuge K, Furuya T, Fujita S, et al.  Lactacystin, a novel microbial metabolite, induces neuritogenesis of neuroblastoma cells. J Antibiot (Tokyo). 1991;44:113–118
  43. Fenteany G, Standaert RF, Lane WS, Choi S, Corey EJ, Schreiber SL. Inhibition of proteasome activities and subunit-specific amino-terminal threonine modification by lactacystin. Science. 1995;268:726–731
  44. Dick LR, Cruikshank AA, Grenier L, Melandri FD, Nunes SL, Stein RL. Mechanistic studies on the inactivation of the proteasome by lactacystin. J Biol Chem. 1996;271:7273–7276
  45. Dick LR, Cruikshank AA, Destree AT, Grenier L, McCormack TA, Melandri FD, et al.  Mechanistic studies on the inactivation of the proteasome by lactacystin in cultured cells. J Biol Chem. 1997;272:182–188
  46. Soucy F, Plamondon L, Behnke M, Roush W. Synthesis of clasto-lactacystin β-lactone and analogs thereof. 1999; USA Application No. 60/055,848
  47. Barnes PJ, Adcock IM. NF-κB: a pivotal role in asthma and a new target for therapy. Trends Pharmacol Sci. 1997;18:46–50
  48. Stein RL, Melandri F, Dick L. Kinetic characterization of the chymotryptic activity of the 20S proteasome. Biochem. 1996;35:3899–3908
  49. Thorne PS, Hawk C, Kaliszewski SD, Guiney PD. The noninvasive mouse ear swelling assay. Fundam Appl Toxicol. 1991;17:790–806
  50. Cannon WC, Blanton EF, McDonald KE. The flow past chamber: an improved nose only exposure system for rodents. Am Indust Hyg Assoc J. 1983;44:923–928
  51. Van Loveren H, Meade R, Askenase PW. An early component of delayed-type hypersensitivity mediated by T cells and mast cells. J Exp Med. 1983;157:1604–1617
  52. Asherson GL, Ptak W. Contact and delayed hypersensitivity in the mouse. I. Active sensitization and passive transfer. Immunology. 1968;15:405–416
  53. Phanuphak P, Moorhead JW, Claman HN. Tolerance and contact sensitivity to DNFB in mice. I. In vivo detection by ear swelling and correlation with in vitro cell stimulation. J Immunol. 1974;112:115–123
  54. Claman HN, Miller SD. Immunoregulation of contact sensitivity. J Invest Dermatol. 1980;74:263–266
  55. Cobb RR, Felts KA, Parry GCN, Mackman N. Proteasome inhibitors block VCAM-1 and ICAM-1 gene expression in endothelial cells without affecting nuclear translocation of nuclear factor-κB. Eur J Immunol. 1996;26:839–845
  56. Butcher EC. Leukocyte-endothelial cell recognition: three (or more) steps to specificity and diversity. Cell. 1991;67:1033–1036
  57. Katz AM, Rosenthal D, Sauder DN. Cell adhesion molecules: structure, function, and implication in a variety of cutaneous and other pathologic conditions. Int J Dermatol. 1991;30:153–160
  58. Picker LJ, Butcher EC. Physiological and molecular mechanisms of lymphocyte homing. Ann Rev Immunol. 1992;10:561–591
  59. Palombella VJ, Conner EM, Fuseler JW, Destree A, Davis JM, Laroux FS, et al.  Role of the proteasome and NF-κB in streptococcal cell wall-induced polyarthritis. Proc Natl Acad Sci. 1998;95:15671–15676
  60. Teelucksingh S, Padfield PL, Tibi L, Gough KJ, Holt PR. Inhaled corticosteroids, bone formation, and osteocalcin. Lancet. 1991;33:60–61
  61. Geddes DM. Inhaled corticosteroids: benefits and risks. Thorax. 1992;47:404–407
  62. Ray A, LaForge S, Sehgal PB. On the mechanism for efficient repression of the interleukin-6 promoter by glucocorticoids: enhancer, TATA box, and RNA start site (Inr motif) occlusion. Mol Cell Biol. 1990;10:5736–5746
  63. Mukaida N, Morita M, Ishikawa Y, Rice N, Okamoto S-I, Kasahara T, et al.  Novel mechanism of glucocorticoid-mediated gene repression. J Biol Chem. 1994;269:13289–13295
  64. Chomczynski P, Qasba P, Topper YJ. Transcriptional and post-transcriptional roles of glucocorticoid in the expression of the rat 25,000 molecular weight casein gene. Biochem Biophys Res Commun. 1986;134:812–818
  65. Auphan N, DiDonato JA, Rosette C, Helmberg A, Karin M. Immunosuppression by glucocorticoids: inhibition of NF-κB activity through induction of IκB synthesis. Science. 1995;270:286–290
  66. Scheinman RI, Cogswell PC, Lofquist AK, Baldwin AS. Role of transcriptional activation of IκBα in mediation of immunosuppression by glucocorticoids. Science. 1995;270:283–286
  67. Hass R, Brach M, Gunji H, Kharbanda S, Kufe D. Inhibition of EGR-1 and NF-κB gene expression by dexamethasone during phorbol ester-induced human monocytic differentiation. Biochem Pharmacol. 1992;44:1569–1576
  68. Ray A, Prefontaine KE. Physical association and functional antagonism between the p65 subunit of transcription factor NF-κB and the glucocorticoid receptor. Proc Natl Acad Sci. 1994;91:752–756
  69. Scheinman RI, Gualberto A, Jewell CM, Cidlowski JA, Baldwin AS. Characterization of mechanism involved in transrepression of NF-κB by activated glucocorticoid receptors. Mol Cell Biol. 1995;15:943–953
  70. Unlap T, Jope RS. Inhibition of NFκB DNA binding activity by glucocorticoids in rat brain. Neurosci Lett. 1995;198:41–44
  71. Pates PC, Ledger PW, Aston R. Recent advances in the treatment of septic shock. Expert Opinion in Therapeutic Patents. 1994;4:917–930

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The Journal of Allergy and Clinical Immunology
Volume 104, Issue 2 , Pages 294-300, August 1999

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