p38 inhibition and not MK2 inhibition enhances the secretion of chemokines from TNF-α activated rheumatoid arthritis fibroblast-like synoviocytes.

OBJECTIVES: For many years the p38 MAP kinase (MAPK) has been a major anti-inflammatory target for the development of an oral therapy for rheumatoid arthritis (RA). However, disappointing results from Phase II clinical studies suggest that adaptations may occur, which allow escape from blockade of the p38 pathway. In this study we investigated whether p38 inhibition mediated JNK activation represents such an escape mechanism. METHODS: Interaction between the JNK and p38 pathways was studied in TNF-α stimulated THP-1 monocytes, primary macrophages and fibroblast-like synoviocytes from OA and RA patients using pharmacological inhibitors and siRNAs. RESULTS: TNF-α induced phosphorylation of JNK and c-Jun was sustained by p38 inhibitors in monocytes, primary macrophages and FLS. Upregulation of Mip1α, Mip1ß and IL-8 mRNAs and protein were observed upon p38 inhibition. More importantly, inhibition of MK2, the substrate of p38 did not sustain JNK activation upon TNF-α activation and did not elevate Mip1α, Mip1ß and IL-8 chemokines as compared to TNF-α alone. In this study, TNF-α or IL-1ß induced JNK activation is sustained by p38 inhibition, resulting in enhanced chemokine secretion. CONCLUSIONS: Based on the suggested role of these chemokines in RA pathogenesis, the upregulation of these chemokines may provide an explanation for the lack of efficacy of p38 inhibitors in Phase II. The absence of any effect of MK2 inhibition in our models on this mechanism, while coming with similar efficacy on blocking p38, provides support for further investigations to reveal the potential of MK2 inhibition as a novel treatment of RA.

CTLA4-IgG reverses asthma manifestations in a mild but not in a more "severe" ongoing murine model.

We investigated whether CTLA4-Ig can reverse established asthma manifestations in a novel murine model of ongoing disease. In BALB/c mice, sensitized to ovalbumin (OVA) without adjuvant, airway inflammation was induced by a first series of OVA aerosol challenges. Murine CTLA4-IgG was then administered, followed by a second series of OVA inhalations. In control-treated mice, two series of OVA challenges induced upregulation of OVA-specific IgE in serum, eosinophils in the bronchoalveolar lavage fluid (BALF), and IL-5 production by lung lymphocytes upon OVA restimulation in vitro, compared with saline-challenged mice. CTLA4-IgG significantly inhibited all of these parameters in OVA-challenged mice. Importantly, mCTLA4-IgG performed better than the gold-standard dexamethasone because this corticosteroid did not inhibit the upregulation of OVA-specific IgE in serum. In a more "severe" ongoing model, induced by sensitization to OVA emulsified in aluminum hydroxide, resulting in airway hyperresponsiveness to methacholine and stronger inflammatory responses, mCTLA4-IgG was less effective in that only the number of eosinophils in the BALF was reduced (P = 0.053), whereas dexamethasone inhibited both BALF eosinophilia and cytokine production by lung lymphocytes. Thus, CTLA4-Ig might be an effective alternative therapy in established allergic asthma, especially in situations of mild disease.

Cloning and pharmacological characterization of a fourth histamine receptor (H(4)) expressed in bone marrow.

Histamine is a multifunctional hormone that regulates smooth muscle contraction in the airways, acid secretion in the gut, and neurotransmitter release in the central nervous system through three well characterized receptor subtypes, H(1), H(2), H(3), respectively. As part of a directed effort to discover novel G-protein-coupled receptors through homology searching of genomic databases, we identified a partial clone (GPCR105) that had significant homology to the recently identified histamine H(3) receptor cDNA. Expression of the full-length human GPCR105 in cells confers the ability to bind [(3)H]histamine with high affinity (K(D) = 5 nM). GPCR105 is pharmacologically similar to the histamine H(3) receptor in that it binds many of the known H(3) agonists and antagonists, albeit with a different rank order of affinity/potency. GPCR105 does not bind (i.e., K(D) > 10 microM) all tested H(1) and H(2) receptor antagonists such as diphenhydramine, loratadine, ranitidine, and cimetidine, but has modest affinity for the H(2) receptor agonist, dimaprit (377 nM). Whereas the H(3) receptor is expressed almost exclusively in nervous tissues, GPRC105 is expressed primarily in bone marrow and eosinophils. Together, these data demonstrate that GPCR105 is a novel histamine receptor structurally and pharmacologically related to the H(3) receptor. However, its unique expression profile and physiological role suggest that GPCR105 is a fourth histamine receptor subtype (H(4)) and may be a therapeutic target for the regulation of immune function, particularly with respect to allergy and asthma.

Tbt-1, a new T-box transcription factor induced in activated Th1 and CD8+ T cells.

Differentiation of CD4+ T cells into T helper (Th) 1 or Th2 cells requires the cytokines interleukin (IL)-12 and IL-4, respectively. However, transcription factors that regulate expression of Th1 or Th2 cell-specific genes remain largely unclear. In the present study, a new Th1-specific transcription factor, named Tbt-1 (T-box protein expressed in T lymphocytes), was identified. Tbt-1 is a novel member of the T-box family, which is characterized by a conserved T-box DNA-binding domain. Unlike other known T-box proteins that regulate embryo development and organogenesis, Tbt-1 expression is restricted to adult lymphoid organs. Tbt-1 mRNA is only detected in peripheral lymphoid tissues such as spleen, lymph nodes, and blood leukocytes, but not in thymus or bone marrow. Tbt-1 mRNA is not detected in resting T cells but is strongly induced in differentiating Thl cells and CD8+ cytotoxic effector cells. In contrast, Tbt-1 expression was not observed in the entire process of Th2 cell differentiation. In addition, phylogenetic analyses indicate that Tbt-1 co-evolved with adaptive immune responses. Thus, Tbt-1 is the first T-box transcription factor shown to be specific for Th1 cell differentiation.

Antigen-stimulated lung CD4+ cells produce IL-5, while lymph node CD4+ cells produce Th2 cytokines concomitant with airway eosinophilia and hyperresponsiveness.

OBJECTIVE AND DESIGN: We investigated whether airway inflammation in a mouse model of allergic asthma is related to antigen-specific T cell responses in the effector organ, the lung, and in the lung draining lymph nodes (LN). MATERIALS AND SUBJECTS: In BALB/c mice pathophysiological parameters were measured in vivo, and lung draining LN and lung cells were restimulated in vitro. TREATMENT: Mice were sensitized with ovalbumin and repeatedly challenged with ovalbumin or saline inhalation. METHODS: Airway reactivity, inflammation in the airways, serum levels of IgE were measured, and cytokine levels and proliferative responses were determined in antigen-stimulated lymphocyte cultures. RESULTS AND CONCLUSIONS: Sensitization results in antigen-specific Th0-like LN cells, despite the presence of antigen-specific IgE. Repeated antigen inhalation induced airway hyperresponsiveness and eosinophil infiltration concomitant with a shift towards Th2 cytokine production exclusively by lung draining LN T cells. Furthermore, these airway symptoms are associated with antigen-specific CD4+ effector T cells in the airway tissue producing only IL-5, but not IL-4, which are unable to proliferate.

Differential effects of endogenous and exogenous interferon-gamma on immunoglobulin E, cellular infiltration, and airway responsiveness in a murine model of allergic asthma.

The inflammatory response as seen in human allergic asthma is thought to be regulated by Th2 cells. It has been shown that interferon-gamma (IFN-gamma) can downregulate the proliferation of Th2 cells and therefore might be of therapeutic use. In the present study we have investigated the in vivo role of endogenous and exogenous IFN-gamma in a murine model with features reminiscent of human allergic asthma. IFN-gamma gene knockout (GKO) and wild-type mice were sensitized with ovalbumin and exposed to repeated ovalbumin aerosol challenges. In addition, wild-type mice were treated with intraperitoneal or nebulized recombinant murine IFN-gamma during the challenge period. Sensitized wild-type mice exhibited upregulated ovalbumin-specific IgE in serum, and airway hyperresponsiveness and infiltration of eosinophils and mononuclear cells in the bronchoalveolar lavage fluid (BALF) after ovalbumin challenge. In contrast, in GKO mice only reduced eosinophilic infiltration in the BALF was observed after ovalbumin challenge. In wild-type mice, parenteral IFN-gamma treatment downregulated ovalbumin-specific IgE levels in serum, and airway hyperresponsiveness and cellular infiltration in the BALF, whereas aerosolized IFN-gamma treatment only suppressed airway hyperresponsiveness. In vitro experiments showed that these effects of IFN-gamma appear not to be mediated via a direct effect on the cytokine production of antigen-specific Th2 cells. These data indicate that airway hyperresponsiveness can be downregulated by IFN-gamma locally in the airways, whereas for downregulation of IgE and cellular infiltration systemic IFN-gamma is needed. The present study shows that exogenous IFN-gamma can downregulate the allergic response via an antigen-specific T-cell independent mechanism, but at the same time endogenous IFN-gamma plays a role in an optimal response.

Allergen immunotherapy inhibits airway eosinophilia and hyperresponsiveness associated with decreased IL-4 production by lymphocytes in a murine model of allergic asthma.

In the present study, we investigated whether allergen immunotherapy is effective in a murine model with immunologic and pathophysiologic features reminiscent of allergic asthma. Ovalbumin-sensitized mice received increasing (1 microgram to 1 mg) subcutaneous doses of ovalbumin twice a week for 8 wk according to a semirush immunotherapy protocol as used in allergic patients. During immunotherapy, an initial rise in serum levels of ovalbumin-specific antibodies (immunoglobulin [Ig]G1, IgE, IgG2a) occurred, after which IgE levels decreased sharply concomitant with an increase in IgG2a levels. The increase in IgG2a levels, with the decline in IgE levels, suggests that during immunotherapy interferon-gamma production is increased or interleukin (IL)-4 production is decreased. After immunotherapy, inhalation challenge of the mice with ovalbumin revealed almost complete inhibition (98%, P < 0.01) of eosinophil infiltration into bronchoalveolar lavage and airway hyperresponsiveness (100% at 320 microgram/kg methacholine, P < 0.05) compared with sham-treated animals. In addition, IL-4 production of thoracic lymph node cells stimulated with ovalbumin in vitro was largely reduced (60%, P < 0.05) after immunotherapy. Thus, effective immunotherapy in this animal model appears to be due to modulation of antigen-specific T cells. Similar effects on airway symptoms and IL-4 production can be obtained within 1 wk by three injections of the highest dose of ovalbumin (1 mg). This animal model will be used as a preclinical model to improve allergen immunotherapy and to gain more insight into the mechanisms involved.

Prevention of Th2-like cell responses by coadministration of IL-12 and IL-18 is associated with inhibition of antigen-induced airway hyperresponsiveness, eosinophilia, and serum IgE levels.

Allergic asthma is thought to be regulated by Th2 cells, and inhibiting this response is a promising mode of intervention. Many studies have focused on differentiation of Th cells to the Th1 or Th2 subset in vitro. IL-4 is essential for Th2 development, while IL-12 induces Th1 development, which can be enhanced by IL-18. In the present study, we investigated whether IL-12 and IL-18 were able to interfere in Th2 development and the associated airway symptoms in a mouse model of allergic asthma. Mice were sensitized with OVA using a protocol that induces IgE production. Repeated challenges by OVA inhalation induced elevated serum levels of IgE, airway hyperresponsiveness, and a predominantly eosinophilic infiltrate in the bronchoalveolar lavage concomitant with the appearance of Ag-specific Th2-like cells in lung tissue and lung-draining lymph nodes. Whereas treatments with neither IL-12 nor IL-18 during the challenge period were effective, combined treatment of IL-12 and IL-18 inhibited Ag-specific Th2-like cell development. This inhibition was associated with an absence of IgE up-regulation, airway hyperresponsiveness, and cellular infiltration in the lavage. These data show that, in vivo, the synergistic action of IL-12 and IL-18 is necessary to prevent Th2-like cell differentiation, and consequently inhibits the development of airway symptoms in a mouse model of allergic asthma.

Vbeta8+ T lymphocytes are essential in the regulation of airway hyperresponsiveness and bronchoalveolar eosinophilia but not in allergen-specific IgE in a murine model of allergic asthma.

BACKGROUND: There is increasing evidence that in allergic asthma the inflammatory process is regulated by T lymphocytes. In BALB/c mice the majority of ovalbumin responsive T lymphocytes express the Vbeta8.1+ and Vbeta8.2+ T-cell receptor. OBJECTIVE: We analysed the contribution of Vbeta8+ T lymphocytes during the sensitization and challenge phase in the regulation of antigen-specific IgE, airway hyperresponsiveness and cellular infiltration in the airways in a murine model of allergic asthma. METHODS: Mice strains genetically lacking (SJL/J and SJA/9) and expressing (BALB/c) the Vbeta8+ T cell receptor were used. In addition, prior to the sensitization and prior to the challenge BALB/c mice were treated with antibodies to Vbeta8. Mice were sensitized with ovalbumin, followed by repeated challenge with ovalbumin or saline aerosols. RESULTS: In ovalbumin challenged BALB/c mice treated with control antibody a significant increase in eosinophils in the bronchoalveolar lavage, airway hyperresponsiveness and increased serum levels of ovalbumin-specific IgE were observed compared to control mice. Treatment of BALB/c mice with antibodies to Vbeta8 prior to the sensitization or prior to the challenge period completely inhibited the ovalbumin induced infiltration of eosinophils and airway hyperresponsiveness, while ovalbumin-specific IgE was slightly decreased. In SJA/9 and SJL/J mice ovalbumin challenge did not induce eosinophilic infiltration and airway hyperresponsiveness. In SJL/J mice ovalbumin challenge induced an upregulation of ovalbumin-specific IgE, however, in SJA/9 mice no upregulation was observed. CONCLUSION: It is demonstrated that Vbeta8+ T lymphocytes are essential for infiltration of eosinophils in the airways and development of airway hyperresponsiveness in a murine model of allergic asthma. In contrast, although Vbeta8+ T lymphocytes seem to be important for the extent of IgE levels, no essential role for Vbeta8+ T lymphocytes in the induction of antigen-specific IgE was observed.

Murine CTLA4-IgG treatment inhibits airway eosinophilia and hyperresponsiveness and attenuates IgE upregulation in a murine model of allergic asthma.

Antigen-specific T-cell activation requires the engagement of the T-cell receptor (TCR) with antigen as well as the engagement of appropriate costimulatory molecules. One of the most important pathways of costimulation is the interaction of CD28 on the T cell with B7-1/B7-2 on antigen-presenting cells. In the present study, we have examined the in vivo effects of blocking the CD28:B7 T-cell costimulatory pathway by administration of mCTLA4-IgG in a murine model of allergic asthma. Mice were sensitized with ovalbumin and exposed to repeated ovalbumin inhalation challenges. In mice treated with a control antibody at the time of ovalbumin challenge a significant increase in the number of eosinophils (12.8 +/- 4.3 x 10(3) cells, P < 0.05) in the bronchoalveolar lavage (BAL) fluid and airway hyperresponsiveness to methacholine (49 +/- 15%, P < 0.05) was observed. In addition, serum levels of ovalbumin-specific IgE were significantly (P < 0.01) increased after ovalbumin challenge compared with saline challenge (1,133 +/- 261 experimental units [EU]/ml and 220 +/- 63 EU/ml, respectively). In mice treated with mCTLA4-IgG at the time of ovalbumin challenge, the infiltration of eosinophils into BAL fluid and the development of airway hyperresponsiveness to methacholine were completely inhibited. The upregulation of ovalbumin-specific IgE levels in serum was attenuated by mCTLA4-IgG treatment. Furthermore, addition of mCTLA4-IgG to cultures of parabronchial lymph node cells from sensitized mice inhibited the ovalbumin-induced interleukin-4 production. These data indicate the therapeutic potential of blocking T-lymphocyte costimulation by CTLA4-IgG as a possible immunosuppressive treatment for patients with allergic asthma.

Bronchoconstriction and airway hyperresponsiveness after ovalbumin inhalation in sensitized mice.

To investigate the mechanisms underlying airway hyperresponsiveness a murine model was developed with several important characteristics of human allergic asthma. Mice were intraperitoneally sensitized with ovalbumin and after 4 weeks challenge via an ovalbumin aerosol. After aerosol, lung function was evaluated with a non-invasive forced oscillation technique. The amount of mucosal exudation into the airway lumen and the presence of mast cell degranulation was determined. Tracheal responsiveness was measured at several time points after challenge. At these time points also bronchoalveolar lavage and histology were performed. Sensitization induced high antigen-specific IgE levels in serum. Inhalation of ovalbumin in sensitized mice induced an immediate but no late bronchoconstrictive response. During this immediate phase, respiratory resistance was increased (54%). Within the first hour after ovalbumin inhalation increased mucosal exudation and mast cell degranulation were observed. At 12 and 24 h after ovalbumin challenge, mice showed tracheal hyperresponsiveness (29% and 34%, respectively). However, no apparent inflammation was found in the lungs or bronchoalveolar lavage. From these results it can be concluded that hyperresponsiveness can develop via mechanisms independent of an inflammatory infiltrate. Since mast cell degranulation occurred after ovalbumin exposure, we hypothesize that mast cells are involved in the induction of airway hyperresponsiveness in this model.