Kinetics of airway hyperresponsiveness and airway eosinophilia in BALB/c mice and their modulation by different dexamethasone treatment regimens.

The mechanisms of airway hyperresponsiveness (AHR) are still poorly understood. In this study we have established a model of persistent AHR and eosinophilia and evaluated the prophylactic vs. therapeutic effects of dexamethasone on these parameters. Mice were immunised with ovalbumin (OVA) on day 0 and challenged intranasally on days 10, 11, 12 and 13 with OVA or phosphate buffer saline (PBS). Airway responsiveness to methacholine, measured 24-h post multiple intranasal OVA challenges, was significantly increased compared to time matched PBS-controls (P<0.05). AHR could be detected for up to 14 days after the last OVA challenge although the magnitude of the AHR had diminished by day 14 compared to day 1. OVA challenge of mice induced a significant airway eosinophilia at 24h (P<0.05); this persisted for 2 weeks after the challenge. Prophylactic treatment with dexamethasone (1mg x kg (-1)) reduced the OVA induced AHR, eosinophilia and mucus cell hyperplasia/metaplasia measured 24h post challenge. Therapeutic treatment, with dexamethasone (2 mg x kg(-1)), significantly inhibited established airway eosinophilia, measured at 72 h post OVA challenge, only when treatment was initiated at 24h but not 48 h after challenge. In contrast, AHR measured at 72 h post OVA challenge was significantly reduced when treatment was started at either 24 or 48 h post challenge. Our data shows that the immunization and challenge procedures employed resulted in a persistent type of AHR. Prophylactic intervention with steroids almost completely inhibited its development; however therapeutic intervention only partially resolved AHR.

New trends in immunotherapy to prevent atopic diseases.

Advances in the understanding of the molecular and cellular immunological mechanism of atopy have led to the development of new therapies for allergic diseases. However, only a few new drugs have reached the clinic and none provides long-term immunomodulatory effects. Immunotherapy, through its capacity to produce a long-term, antigen-specific, protective immune response, is the only aetiologic treatment that offers the possibility of preventing or even curing atopic diseases. However, the potential severe side-effects associated with conventional immunotherapy using whole allergen extract limits its widespread use. Thus, novel strategies to minimize the side-effects and improve the efficacy of immunotherapy are of considerable interest in the treatment of atopic diseases. Promising animal and human studies, using approaches such as peptide immunotherapy, DNA vaccination, CpG oligonucleotides and mycobacterial vaccines, suggest that it might be possible to prevent or cure atopic diseases in the future.

Requirement for gammadelta T cells in allergic airway inflammation.

The factors that contribute to allergic asthma are unclear but the resulting condition is considered a consequence of a type-2 T helper (TH2) cell response. In a model of pulmonary allergic inflammation, mice that lacked gammadelta T cells had decreases in specific immunoglobulin E (IgE) and IgG1 and pulmonary interleukin-5 (IL-5) release as well as in eosinophil and T cell infiltration compared with wild-type mice. These responses were restored by administration of IL-4 to gammadelta T cell-deficient mice during the primary immunization. Thus, gammadelta T cells are essential for inducing IL-4-dependent IgE and IgG1 responses and for TH2-mediated airway inflammation to peptidic antigens.

Inhibition by the immunosuppressive agent FK-506 of antigen-induced airways eosinophilia and bronchial hyperreactivity in mice.

1. The effect of the immunosuppressive agent, FK-506, an allergen-induced airways eosinophilia and bronchial hyperreactivity (BHR) in hyper IgE mice (BP2 selection) was investigated. 2. Administration of FK-506 at 2 mg kg-1 s.c., 1 h before and 5 h after the first four ovalbumin challenges, reduced the recruitment of eosinophils into the bronchoalveolar lavage fluid (BALF) from 1.36 +/- 0.22 x 10(5) to 0.53 +/- 0.24 x 10(5) cells ml-1 (n = 5-6, P < 0.05; 60% inhibition), inhibited by 80% BHR in response to i.v. 5-HT and practically suppressed BHR in response to inhaled methacholine. 3. The antigen-induced interleukin (IL)-5 formation in the BALF and serum was inhibited by FK-506 by 75% in both instances. 4. FK-506 failed to modify the bronchoconstriction in BP2 mice, suggesting that different mechanisms are involved in acute bronchoconstriction and BHR. 5. The increased number of CD4+, CD8+, CD3+ T lymphocytes in the BALF to antigen-challenged mice was unaffected by FK-506. 6. These findings indicate that antigen-induced in vivo IL-5 release and eosinophil, but not T-cell, infiltration into the bronchial lumen of sensitized BP2 mice are targets for the anti-allergic activities of FK-506.

Modulation by IL-10 of antigen-induced allergic responses in mice.

Over the last few years, we examined the anti-allergic properties of interleukin (IL)-10 in different models of inflammation in the mouse, as well as against IgE-dependent activation of mouse bone marrow-derived mast cells (BMMC). We showed that IL-10, concurrently administered with ovalbumin, inhibited inflammatory cell accumulation in the airways and in the peritoneal cavity of sensitized mice, as well as the accompanying cytokine release. IL-10 also blocked antigen-induced cytokine generation by IgE-stimulated BMMC. Together, these results identify a novel biological property of IL-10, as a cytokine with potent anti-allergic activities.

Modulation by IL-10 of antigen-induced IL-5 generation, and CD4+ T lymphocyte and eosinophil infiltration into the mouse peritoneal cavity.

Sensitized BALB/c mice challenged i.p. with 1 microgram of OVA showed IL-5 release in the peritoneal lavage fluid, which peaked at 6 h and decreased thereafter. This was followed by a massive eosinophil accumulation, which started at 6 h and reached a plateau between 24 and 48 h. The i.p. injection of recombinant murine (rm) IL-10 (0.01-0.1 microgram/cavity) along with OVA reduced IL-5 release at 6 h and allergic eosinophilia at 6, 24, and 48 h. rmIL-10 also blocked in vitro IL-5 generation by sensitized peritoneal cells cultured in the presence of OVA. The inhibitory effect of rmIL-10 on Ag-induced eosinophilia and IL-5 release was suppressed by pretreatment of the animals with 1 mg/mouse of a neutralizing anti-mIL-10 mAb. Flow cytometric analysis revealed an increase in the number of CD4+ and CD8+ T lymphocytes and in the number of CD25+/CD4+ cells in the peritoneal lavage fluid collected 24 and 48 h after challenge, respectively; these numbers were reduced significantly by the administration of 0.1 microgram of rmIL-10. Finally, rmIL-10 failed to modify the anti-CD3-induced IL-5 release in vivo in the peritoneal cavity and in vitro from purified spleen CD4+ T lymphocytes. This suggests that rmIL-10 acts indirectly, by deactivating APC, rather than directly on T cell activation. These findings indicate that rmIL-10 displays anti-allergic activity in sensitized BALB/c mice by preventing Ag-induced CD4+ T lymphocyte and eosinophil accumulation as well as IL-5 release in the peritoneal cavity.

Interleukin-10 inhibits cytokine generation from mast cells.

This report examines the effects of recombinant murine interleukin-10 (rmIL-10) on antigen-induced beta-hexosaminidase, leukotriene (LT)C4 and cytokine release from mouse bone marrow-derived mast cells (BMMC). BMMC sensitized to hapten-monoclonal IgE directed against dinitrophenol-bovine serum albumin (DNP-BSA) and challenged with 10 ng/ml DNP-BSA generated beta-hexosaminidase and LTC4-like material which was followed by tumor necrosis factor-alpha (TNF-alpha) and granulocyte-macrophage colony-stimulating factor (GM-CSF) mRNA expression and protein release. Incubation of BMMC with 1-100 ng/ml rmIL-10 inhibited cytokine generation, without affecting beta-hexosaminidase and LTC4-like material release. TNF-alpha, but not GM-CSF mRNA expression, was also diminished in rmIL-10-treated BMMC, suggesting that down-regulation of cytokine production by rmIL-10 involves different mechanisms. These results identify a novel biological action of IL-10 as an inhibitor of cytokine production by stimulated mast cells.

Interleukin-10 inhibits antigen-induced cellular recruitment into the airways of sensitized mice.

This report examines the effect of recombinant murine (rm) IL-10 on antigen-induced cellular recruitment into the airways of sensitized Balb/c mice. The intranasal instillation of 10 micrograms ovalbumin induced an early (6-24 h) increase in the number of neutrophils, and a late rise (24-96 h) in that of eosinophils in the bronchoalveolar lavage (BAL) fluid and bronchial tissue. A single intranasal instillation of 0.01-0.1 microgram of rmIL-10, administered concurrently with ovalbumin, but not 1 or 3 h thereafter, dose-dependently inhibited both airway neutrophilia and eosinophilia. This phenomenon was suppressed by treating the sensitized mice with 1 mg/mouse of a neutralizing anti-IL-10 mAb, which increased significantly ovalbumin-induced neutrophil and eosinophil accumulation in the BAL fluid. These results suggest that antigen stimulation may trigger the in vivo generation of IL-10, which, in turn, participates in the leukocyte infiltration into the airways. rmIL-10 also reduced TNF-alpha release in the BAL fluid observed 1 and 3 h after antigen challenge. Furthermore, the intranasal instillation of an anti-TNF-alpha antiserum to sensitized mice markedly reduced ovalbumin-induced neutrophil and eosinophil accumulation in the BAL fluid. These findings indicate that leukocyte infiltration into the airways of antigen-challenged mice is regulated by IL-10. Furthermore, inhibition of TNF-alpha production by rmIL-10 suggests that allergic airway inflammation and TNF-alpha formation are parallel events in this model.

Strain-dependency of leukotriene C4 generation from isolated lungs of immunized mice.

1. The antigen-induced leukotriene C4 (LTC4)-like-material release from isolated perfused lungs of actively sensitized Swiss, Balb/C and CBA/J mice was compared. The intra-tracheal (i.t.) instillation of 1 and 100 micrograms ovalbumin to lungs from Swiss mice was followed by a dose-dependent generation of LTC4-like material into the effluent, as detected by radio-immunoassay and h.p.l.c., followed by an enzyme-immunoassay. In contrast, lungs from sensitized Balb/C and CBA/J mice failed to exhibit LTC4-like-material release in amounts above the basal values. No histamine secretion was observed when lungs of the three strains of mice were challenged with ovalbumin. 2. The i.t. instillation of 1 or 10 micrograms platelet-activating factor (PAF) or of 100 micrograms arachidonic acid to lungs from non-sensitized mice, induced the release of comparable amounts of LTC4-like-material in the effluent, irrespective to the strain. However, N-formyl-L-methionyl-L-leucyl-L- phenylalanine (fMLP, 0.1, 10 micrograms), was more effective in inducing the release of LTC4-like-material from lung from Swiss and CBA, than from Balb/C, mice. 3. The intraplantar injection of 0.01 microgram ovalbumin to sensitized Swiss mice induced an intense oedema formation, as measured plethysmographically, while Balb/C mice required a dose of antigen at least 10 fold higher for a similar response. CBA/J mice did not respond to antigen challenge in terms of oedema formation. The intraplantar injection of PAF or fMLP to non-immunized mice induced an oedema of similar intensity in all the strains considered. Accordingly, the different responses to ovalbumin of the three strains of mice is not accounted for by different paw responsiveness to inflammatory mediators.4. Swiss and CBA/J mice exhibited higher titers of circulating IgG antibodies, as measured by passive cutaneous anaphylaxis (PCA), than Balb/C mice. Conversely, lower IgE titers were measured in the serum of sensitized Swiss and CBA/J mice, as compared to Balb/C.5. Our results demonstrate a strain-dependency of antigen-induced LTC4 release from lungs from sensitized mice. This difference is related to the ability of sensitized animals to develop immediate hypersensitivity responses, such as paw oedema formation, but not to the antibody subclass involved in the immunization. Strain-dependent factors may influence the intensity of the response to antigen stimulation. It is thus essential to characterize the different components of the immune response when mouse models for studying hypersensitivity reactions are developed.

Involvement of leukotrienes in allergic inflammation in mice.

The involvement of leukotrienes in the anaphylactic paw edema was investigated in sensitized boosted or unboosted mice. No difference was noted in the intensity of the antigen-induced paw edema between boosted and unboosted animals. Since the non-steroidal anti-inflammatory agents, indomethacin and aspirin, were inactive, cyclooxygenase products are not involved in the anaphylactic reaction in boosted or unboosted mice. In contrast, the mixed lipoxygenase/cyclooxygenase inhibitor, BW 755C (3-amino-1-m-(trifluoroethyl)phenyl-2-pyrazoline), and the LTD4 (leukotriene D4) receptor antagonist, LY 171883 (1-(2-hydroxy-3-propyl-4-(4-(1H-tetrazol-5-yl)butoxy)phenyl)eth anone, reduced the edema in boosted but not unboosted mice, suggesting the involvement of lipoxygenase metabolites in the allergic edema in boosted mice. Dexamethasone was only effective in inhibiting allergic edema in boosted mice when administered 12 h before provocation, but failed to reduce the edema in unboosted mice. Our results suggest that the booster injection during immunization shifts the anaphylactic mouse paw edema from a leukotriene-independent to a leukotriene-dependent reaction.

Characterization and pharmacological modulation of antigen-induced peritonitis in actively sensitized mice.

1. The intraperitoneal (i.p.) injection of 1 or 10 micrograms ovalbumin to sensitized Balb/c mice led to an acute histamine release, firstly evidenced 1 min after the challenge and returning to basal levels 30 min thereafter. This phenomenon was unaccompanied by protein extravasation. A dose-dependent increase in the amounts of immunoreactive leukotriene (LT) C4 and LTB4 was observed in the peritoneal washing from sensitized mice 6 h after 1 or 10 micrograms ovalbumin administration. In separate experiments, the i.p. administration of 1 mg activated zymosan to non-immunized mice was followed by a marked protein extravasation, and by immunoreactive LTC4 and LTB4, but not histamine, release in mouse peritoneum 1 h after its injection. 2. Mediator release in the mice peritoneal cavity was concomitant with a transient neutrophil infiltration, which peaked at 6 h and returned to basal levels therefore. An intense eosinophil accumulation starting at 24 h, peaking at 48 h and returning to basal values at 164 h, was also observed. 3. Ovalbumin (1 microgram)-induced eosinophilia, observed at 24 h, was reduced by the pretreatment of the animals with dexamethasone (1 mg kg-1, s.c.) or with the 5-lipoxygenase inhibitor, BWA4C (20 mg kg-1, s.c.), whereas indomethacin (2 mg kg-1, s.c.) and the platelet-activating factor (PAF)-antagonist SR 27417 (10 mg kg-1, s.c.) were ineffective. These results indicate that metabolites of arachidonic acid of lipoxygenase pathway, but not cyclo-oxygenase derivatives or PAF, mediate antigen-induced eosinophil accumulation in the mouse peritoneum. 4. The histamine HI receptor antagonist drug, cetirizine (15-30 mg kg-1, s.c.) markedly reduced ovalbumin-induced eosinophil accumulation under conditions where terfenadine was ineffective, suggesting that the effect of cetirizine was not related to the inhibition of the H1 receptor effects of histamine.5. The immunosuppressive agent, FK-506 (1-2 mg kg-1, s.c.) and the protein synthesis inhibitor,cylcoheximide, when administered either in situ (0.06 ng/cavity) or systemically (5 mg kg-1, s.c.),prevented antigen-induced eosinophil accumulation in the mouse peritoneum, contributing to the concept that substances (probably cytokines) originating from lymphocytes may be involved in the modulation of the eosinophilotactic response in this model.6. The results of the present study indicate that the i.p. administration of ovalbumin to actively sensitized mice induced late eosinophil accumulation in the peritoneal cavity. This phenomenon, which may be in part mediated by the release of lipoxygenase metabolites and/or by newly generated factors,such as T-lymphocytes-derived eosinophilotactic cytokines, offers an interesting tool to investigate the mechanism of action of anti-allergic and anti-inflammatory drugs.

Lung responsiveness to antigen in sensitised mice of different strains.

A new model of the isolated perfused lung from different strains of mice was developed. Lungs from Swiss, Balb/C and CBA mice actively sensitised to ovalbumin were challenged intratracheally (i.t.) by antigen on day 14. In Swiss mice instillation of ovalbumin led to the release of leukotriene (LT) C4 significantly above basal values. Conversely, lungs from Balb/C and CBA mice were unresponsive to ovalbumin in terms of production of LTC4. All strains failed to release histamine when challenged with antigen. Intratracheal instillation of platelet-activating factor (PAF), to lungs from non-sensitised animals, induced the release of comparable amounts of LTC4, irrespective of the strain. In contrast, i.t. administration of fMLP to lungs from Swiss mice elicited release of significantly higher amounts of LTC4 as compared to Balb/C and CBA mice. In separate experiments, ovalbumin was injected into the paws and anaphylactic oedema was evaluated. Balb/C and CBA required 1 microgram to show an oedema formation, whereas the dose of ovalbumin for Swiss mice to develop a similar response was at least 30-fold lower. In conclusion, antigen provocation induced release of LTC4 from lungs from Swiss mice but not from Balb/C or CBA. This difference may be accounted for by strain-dependent factors, such as antibody production and requires further investigation.