Role of HLA-G as a predictive marker of low risk of chronic rejection in lung transplant recipients: a clinical prospective study.

Human leukocyte antigen G (HLA-G) expression is thought to be associated with a tolerance state following solid organ transplantation. In a lung transplant (LTx) recipient cohort, we assessed (1) the role of HLA-G expression as a predictor of graft acceptance, and (2) the relationship between (i) graft and peripheral HLA-G expression, (ii) HLA-G expression and humoral immunity and (iii) HLA-G expression and lung microenvironment. We prospectively enrolled 63 LTx recipients (median follow-up 3.26 years [min: 0.44-max: 5.03]). At 3 and 12 months post-LTx, we analyzed graft HLA-G expression by immunohistochemistry, plasma soluble HLA-G (sHLA-G) level by enzyme-linked immunosorbent assay, bronchoalveolar lavage fluid (BALF) levels of cytokines involved in chronic lung allograft dysfunction (CLAD) and anti-HLA antibodies (Abs) in serum. In a time-dependent Cox model, lung HLA-G expression had a protective effect on CLAD occurrence (hazard ratio: 0.13 [0.03-0.58]; p = 0.008). The same results were found when computing 3-month and 1-year conditional freedom from CLAD (p = 0.03 and 0.04, respectively [log-rank test]). Presence of anti-HLA Abs was inversely associated with graft HLA-G expression (p = 0.02). Increased BALF level of transforming growth factor-ß was associated with high plasma sHLA-G level (p = 0.02). In conclusion, early graft HLA-G expression in LTx recipients with a stable condition was associated with graft acceptance in the long term.

Immunohistochemical study of HLA-G expression in lung transplant recipients.

Human leukocyte antigen-G (HLA-G), a nonclassical HLA class I protein, promotes immune tolerance of solid-organ allografts, yet its role in lung transplantation (LTx) is unknown. We examined the expression of HLA-G in lung allografts through immunohistochemistry by a cross-sectional study of 64 LTx recipients, classified into four groups (stable patients, acute rejection [AR], bronchiolitis obliterans syndrome [BOS] and symptomatic viral shedders). A marked expression of HLA-G in bronchial epithelial cells (BEC) was frequently observed in stable recipients (n = 18/35 [51%]), but not in patients with AR (n = 14) or with BOS (n = 8). HLA-G was also expressed by 4 of 7 symptomatic viral shedders. In addition, HLA-G-positive patients from the stable group (n = 35) experienced lower incidence of resistant AR and/or BOS during long-term follow-up, as compared with their HLA-G-negative counterparts. Finally, in vitro data showed that interferon-gamma, a cytokine present in lung allograft microenvironment, upregulated HLA-G mRNA and protein expression in primary cultured human BEC. We conclude that HLA-G expression in the bronchial epithelium of lung allograft is elevated in some LTx recipients in association with their functional stability, suggesting a potential role of HLA-G as a tolerance marker.

Glucocorticoid-induced apoptosis in human eosinophils: mechanisms of action.

Prominent blood and tissue eosinophilia is clinically manifested in a number of inflammatory states, particularly in allergic diseases. Corticosteroids are the most effective anti-inflammatory drugs used in the treatment of eosinophilic disorders, including bronchial asthma. Their beneficial effects result, among others, from (i) the suppression of the synthesis and the effects of eosinophil survival factors, (ii) the direct induction of eosinophil apoptosis and (iii) the stimulation of their engulfment by professional phagocytic cells. Failure of steroids to propagate apoptotic signals and to promote eosinophil clearance may explain the corticoresistance observed in a proportion of asthmatic patients. Accordingly, studies on the intracellular mechanisms involved in eosinophil corticosensitivity and resistance may provide a valuable tool for identifying new and selective molecular targets to therapeutically resolve otherwise persistent eosinophilic inflammation. In this review, the intracellular cascade of events involved in corticosteroid-mediated eosinophil apoptotic death is discussed and compared to the signalling pathway governing this process in the established model of dexamethasone-induced thymocyte apoptosis.

Involvement of caspases and of mitochondria in Fas ligation-induced eosinophil apoptosis: modulation by interleukin-5 and interferon-gamma.

In this study, we examined the relative importance of caspases and mitochondria in Fas-mediated eosinophil apoptosis. Stimulation of human peripheral blood eosinophils with an agonistic anti-human Fas monoclonal antibody, but not with control IgM, induced a time-dependent increase in their apoptosis, which was associated with a loss in mitochondrial transmembrane potential (DeltaPsi(m)) and with caspase-8 and caspase-3 activation. Interleukin (IL)-5 and interferon (IFN)-gamma, two cytokines known to prolong eosinophil survival, inhibited Fas-mediated apoptosis and caspase activation but poorly affected the decrease in DeltaPsi(m). Eosinophil incubation with bongkrekic acid, an inhibitor of the mitochondrial permeability transition pore (MPTP) opening, failed to modify Fas-mediated loss in DeltaPsi(m), caspase activation, and apoptosis. In contrast, caspase inhibitors markedly reduced eosinophil apoptosis without significantly affecting DeltaPsi(m) dissipation. We conclude that caspase-8 and caspase-3 activation, but not MPTP opening, mediate Fas-induced eosinophil apoptosis and are the main targets for the protective effect of IL-5 and IFN-gamma.

Regulation of peroxisome proliferator-activated receptor gamma expression in human asthmatic airways: relationship with proliferation, apoptosis, and airway remodeling.

Airway inflammation and alterations in cellular turnover are histopathologic features of asthma. We show that the expression of peroxisome proliferator-activated receptor gamma (PPAR gamma), a nuclear hormone receptor involved in cell activation, differentiation, proliferation, and/or apoptosis, is augmented in the bronchial submucosa, the airway epithelium, and the smooth muscle of steroid-untreated asthmatics, as compared with control subjects. This is associated with enhanced proliferation and apoptosis of airway epithelial and submucosal cells, as assessed by the immunodetection of the nuclear antigen Ki67, and of the cleaved form of caspase-3, respectively, and with signs of airway remodeling, including thickness of the subepithelial membrane (SBM) and collagen deposition. PPAR gamma expression in the epithelium correlates positively with SBM thickening and collagen deposition, whereas PPAR gamma expressing cells in the submucosa relate both to SBM thickening and to the number of proliferating cells. The intensity of PPAR gamma expression in the bronchial submucosa, the airway epithelium, and the smooth muscle is negatively related to FEV(1) values. Inhaled steroids alone, or associated with oral steroids, downregulate PPAR gamma expression in all the compartments, cell proliferation, SBM thickness, and collagen deposition, whereas they increase apoptotic cell numbers in the epithelium and the submucosa. Our findings have demonstrated that PPAR gamma (1) is a new indicator of airway inflammation and remodeling in asthma; (2) may be involved in extracellular matrix remodeling and submucosal cell proliferation; (3) is a target for steroid therapy.

Decreased pulmonary and tracheal smooth muscle expression and activity of type 1 nitric oxide synthase (nNOS) after ovalbumin immunization and multiple aerosol challenge in guinea pigs.

Pharmacological evidence supports a role of a transient decreased endogenous nitric oxide (NO) synthesis in ovalbumin (OVA)-induced early airway hyperresponsiveness in guinea pigs. However, no data are available regarding the expression and activity of the constitutive NO synthases (cNOS; NOS1 and NOS3, nNOS and eNOS, respectively) in this model. Therefore, we evaluated cNOS activity (conversion of L-[3H]arginine to L-[3H]citrulline in the presence of Ca2+ and calmodulin), nitrate and nitrite (NOx) concentration (modified Griess method), and NOS1 and NOS3 protein expression (Western blot) in lung homogenates and in the tracheal smooth muscle from OVA-immunized and multiple aerosol-challenged guinea pigs (six challenges, once daily). The expression and activity of the inducible NOS isoform (NOS2), the levels of exhaled NO, and the in vivo airway reactivity were also determined. Constitutive NOS activity and NO(x) concentration were significantly lower 6 h after the last OVA challenge as compared with saline exposure, being similar at 24 h. Expression of NOS1 paralleled cNOS activity, which was reduced 6, but not 24 h after OVA challenge. The decrease in NOS1 expression was accompanied by a significant decrease in the amounts of exhaled NO and by a maximal airway hyperresponsiveness to histamine. The levels of NOS3 were not modified at the two time points evaluated, and no NOS2 expression and activity were found at any time point. Similar modifications were observed in the tracheal smooth muscle. We conclude that OVA stimulation in immunized guinea pigs induced a transient reduction in NOS1 protein expression and activity in the respiratory system, which probably participates in airway hyperresponsiveness.

[Eosinophil apoptosis in asthma]. / L'apoptose des éosinophiles dans l'asthme.

Eosinophils play a major role in the onset and maintenance of bronchial inflammation and tissue injury in asthma. Like other leukocytes, eosinophils present in excessive numbers in inflamed tissues are removed by apoptosis. This phenomenon, also called 'programmed cell death', allows elimination of dangerous or redundant cells, thereby ensuring maintenance of tissue homeostasis. It has been suggested that a defect in eosinophil apoptosis would participate in the development and persistence of allergic airways inflammation in asthma. Eosinophil apoptosis, as well as the expression and function of various molecules determining this process, are closely regulated by various stimuli, including cytokines, lipid mediators and growth factors released by various cell types and by the eosinophil itself, as well as exogenous molecules, such as glucocorticoids. These stimuli have been shown to alter the expression and function of different molecules involved in the cascade of events characterising the apoptotic process, particularly Bcl-2 family proteins and the pro-apoptotic membrane glycoprotein, Fas. These observations, together with a better understanding of the mechanisms underlying eosinophil apoptosis, will help to more clearly define the molecular events involved in accumulation of these cells in blood and tissues and to identify potential new targets for the treatment of allergic diseases.

IL-5 and eosinophils mediate the rejection of fully histoincompatible vascularized cardiac allografts: regulatory role of alloreactive CD8(+) T lymphocytes and IFN-gamma.

CD8(+) T lymphocytes are known to inhibit the development of eosinophilia and IL-5 synthesis in models of experimental lung disease. In transplantation, the rejection of fully mismatched cardiac allografts by recipients depleted of CD8(+) T cells is characterized by the recruitment of eosinophils in the rejected organs. We show here that this intragraft eosinophilia is dependent on the production of IL-5 since hearts transplanted into IL-5-deficient recipients depleted of CD8(+) cells did not contain eosinophils. More importantly, allograft survival was significantly extended in these animals. In mixed lymphocyte cultures (MLC), the presence of CD8(+) T cells in the responding cell population inhibited the secretion of IL-5. This inhibition was IFN-gamma dependent since adding neutralizing anti-IFN-gamma antibodies induced the production of IL-5. Furthermore, spleen cells isolated from IFN-gamma receptor (IFN-gammaR)-deficient mice secreted IL-5 upon allogeneic stimulation in primary MLC. In vivo, eosinophilia was observed in allografts rejected by IFN-gammaR-deficient recipients. On the contrary, grafts rejected by IFN-gammaR-deficient mice treated with neutralizing anti-IL-5 antibodies did not exhibit eosinophilic infiltration. Our study reveals the capacity of IL-5-secreting CD4(+) T cells and eosinophils to promote the rejection of heart allograft and demonstrates the importance of CD8(+) T cells and IFN-gamma in regulating this pathway of rejection.

IL-5 mediates eosinophilic rejection of MHC class II-disparate skin allografts in mice.

CD4 T cells play a crucial role in the acute rejection of MHC class II-disparate skin allografts, mainly by Fas/Fas ligand-mediated cytotoxicity. Because recent observations indicate that eosinophils may be found within allografts rejected by CD4 T cells, we evaluated the role played by IL-5, the main eosinophil growth factor, and by eosinophils in the rejection of MHC class II-disparate skin grafts. C57BL/6 mice rapidly rejected MHC class II-disparate bm12 skin grafts. Rejected skins contained a dense, aggressive eosinophil infiltrate. Lymphocytes isolated from lymph nodes draining rejected bm12 skin were primed for IL-5 secretion, and IL-5 mRNA was present within rejected grafts. The IL-5/eosinophil pathway played an effector role in allograft destruction, because the rejection of bm12 skin was significantly delayed in IL-5-deficient mice as compared with wild-type animals. The role of the IL-5/eosinophil pathway was further investigated in MHC class II-disparate donor-recipient strains unable to establish Fas/Fas ligand interactions. Fas ligand-deficient gld/gld mice rejected bm12 skins, and bm12 mice rejected Fas-deficient lpr/lpr C57BL/6 skins. Neutralization of IL-5 prevented acute rejection in both combinations. We conclude that MHC class II-disparate skin allografts trigger an IL-5-dependent infiltration of eosinophils that is sufficient to result in acute graft destruction.

Critical roles for IL-4, IL-5, and eosinophils in chronic skin allograft rejection.

C57BL/6 mice injected with the 145-2C11 anti-CD3 mAb and grafted with MHC class II disparate bm12 skin develop a chronic rejection characterized by interstitial dermal fibrosis, a marked eosinophil infiltrate, and an obliterative intimal vasculopathy. Because these changes occur in the absence of alloreactive antibodies, we examined the contribution of cytokines in their pathogenesis. Chronically rejected grafts showed a marked accumulation of both IL-4 and IL-5 mRNA. Mixed lymphocyte reaction experiments established that mice undergoing chronic rejection were primed for IL-4, IL-5, and IL-10 secretion. In vivo administration of anti-IL-4 mAb completely prevented allograft vasculopathy as well as graft eosinophil infiltration and dermal fibrosis. Injection of anti-IL-5 mAb or the use of IL-5-deficient mice as recipients also resulted in the lack of eosinophil infiltration or dermal fibrosis, but these mice did develop allograft vasculopathy. Administration of anti-IL-10 mAb did not influence any histologic parameter of chronic rejection. Thus, in this model, IL-4- and IL-5-mediated tissue allograft eosinophil infiltration is associated with interstitial fibrosis. IL-4, but not eosinophils, is also required for the development of obliterative graft arteriolopathy.

Prevention of antigen-induced bronchial hyperreactivity and airway inflammation in sensitized guinea-pigs by tacrolimus.

We examined the effect of the immunosuppressive agent, tacrolimus (FK506), on antigen-induced bronchial hyperreactivity to acetylcholine and leukocyte infiltration into the airways of ovalbumin-challenged guinea-pigs. Subcutaneous injection of 0.5 mg/kg of FK506, 1 h before and 5 h after intra-nasal antigen challenge prevented bronchial hyperreactivity to aerosolized acetylcholine, eosinophilia in bronchoalveolar lavage (BAL) fluid and bronchial tissue and the invasion of the bronchial wall by CD4+ T-lymphocytes. FK506 also suppressed ovalbumin-induced increase in the number of leukocytes adhering to the pulmonary vascular endothelium and expressing alpha4-integrins. Inhibition by FK506 of antigen-induced bronchial hyperreactivity in sensitized guinea-pigs may thus relate to its ability to prevent the emergence of important inflammatory components of airway inflammation, such as eosinophil accumulation, as well as CD4+ T-lymphocyte infiltration into the bronchial tissue.

Apoptosis, proliferation, and expression of Bcl-2, Fas, and Fas ligand in bronchial biopsies from asthmatics.

The in situ apoptosis and the expression of molecules involved in this process, such as Bcl-2, Fas, and its ligand, Fas ligand (FasL), were examined in bronchial biopsies from healthy control subjects and from steroid-untreated or -treated asthmatics, using terminal transferase-mediated deoxyuridyltriphosphate nick-end labeling and immunohistochemical techniques, respectively. Bronchial submucosa from steroid- untreated asthmatics showed an increase in the number of eosinophils and a decrease in that of apoptotic cells compared with that of control subjects, but no significant changes in the number of T lymphocytes or in that of cells expressing Bcl-2, Fas, or FasL. Treatment with steroids reduced airway eosinophilia and augmented the proportion of apoptotic eosinophils. Compared with control subjects or untreated patients, steroid-treated asthmatics exhibited increased expression of Bcl-2, Fas, FasL, and of proliferating cell nuclear antigen (PCNA) in their bronchial epithelium, without changes in the number of apoptotic cells. Moreover, the intensity of the expression of Bcl-2, Fas, and FasL correlates well with that of PCNA. We conclude that steroids may reduce the inflammatory cell infiltrate in the bronchial submucosa in part by promoting eosinophil apoptosis and by inducing the expression of FasL on bronchial epithelial cells. Treatment with steroids may also augment survival and proliferation of epithelial cells, possibly via the expression of Bcl-2 and PCNA.

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.

Antihistaminic and antiallergic properties of dextro-mequitamium iodide in upper and lower guinea pig airways: comparison with azelastine.

1. The ability of dextro-mequitamium iodide (d-Meq) to antagonize bronchomotor and inflammatory effects mediated by histamine and antigen challenge in the upper or lower guinea pig airways or both and its potential activity against the recruitment and activation of eosinophils in the bronchial wall have been evaluated in comparison with azelastine. 2. In receptor-binding studies, d-Meq displayed a nanomolar affinity for H1 and muscarinic receptors, and it was endowed with potent bronchodilating properties in the nanomolar range toward tonic contractions induced by histamine and carbachol. 3. d-Meq (100-1,000 nmol/guinea pig) and azelastine (100-5,000 nmol/guinea pig) administered by aerosol significantly inhibited histamine- and antigen-induced increases in insufflation pressure in sensitized animals. 4. d-Meq (1,000-6,000 nmol/kg i.v.) dose dependently inhibited the histamine- or antigen-induced increase in vascular permeability in the upper airways. 5. d-Meq was more effective against histamine than antigen challenge, and its potency was similar or greater than that of azelastine. 6. Aerosolized d-Meq (1,000 nmol/animal) reduced antigen-induced eosinophil accumulation in the bronchoalveolar lavage (BAL) fluid from sensitized guinea pigs. 7. Eosinophils recovered from the BAL fluid of antigen-challenged animals showed an increased chemotaxis in response to LTB4 or platelet-activating factor. Both d-Meq and azelastine (300 nmol/animal) reduced this increase without affecting direct chemotaxis induced by leukotriene B4 (LTB4). 8. These findings provide evidence that local administration of d-Meq might be useful in the treatment of allergic disorders, such as rhinitis and asthma.

Human eosinophils express bcl-2 family proteins: modulation of Mcl-1 expression by IFN-gamma.

The expression of the Bcl-2 family proteins Bax, Mcl-1, Bcl-2, and Bcl-xL, was examined in human peripheral blood eosinophils or in umbilical-cord-blood-derived eosinophils. Immunoblot analysis disclosed high amounts of the proapoptotic factor Bax in freshly purified eosinophils of both types. Although cord-blood-derived eosinophils expressed easily detectable levels of Mcl-1, Bcl-2, and Bcl-xL, only traces or no expression of these three antiapoptotic proteins were found in peripheral blood eosinophils. Incubation of both eosinophil types for 1 to 3 days in a cytokine-deprived medium led to apoptosis, without changes in the expression of Bax, Mcl-1, Bcl-2, or Bcl-xL. Although addition of interleukin-5 or interferon-gamma (IFN-gamma) to the culture medium increased the survival of both eosinophil types, a rise in the levels of Mcl-1 was observed only in IFN-gamma-treated cord-blood eosinophils. Together, these results indicate that human eosinophils have a specific profile of Bcl-2-family protein expression that depends on their maturation status and may be modulated by stimuli that influence their survival.

Role of adenylate cyclase-hemolysin in alveolar macrophage apoptosis during Bordetella pertussis infection in vivo.

Bordetella pertussis induces in vitro apoptosis of murine alveolar macrophages by a mechanism that is dependent on expression of bacterial adenylate cyclase-hemolysin. Using a murine respiratory model, we found in this study that intranasal infection with a parental B. pertussis strain, but not with an isogenic variant deficient in the expression of all toxins and adhesins, induced a marked neutrophil accumulation in the bronchoalveolar lavage fluid and an early decrease in macrophage numbers. These phenomena paralleled a time-dependent rise in the proportion of apoptotic nuclei, as detected by flow cytometry, and of macrophages which had engulfed apoptotic bodies. Apoptotic death of bronchopulmonary cells was observed exclusively following intranasal infection with bacteria reisolated from lungs of infected animals and not with B. pertussis collected after in vitro subculture. Using the terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling technique coupled to fluorescence microscopy and morphological analysis, we established that the apoptotic cells in bronchoalveolar lavage fluids were neutrophils and macrophages. Histological analysis of the lung tissues from B. pertussis-infected mice showed increased numbers of apoptotic cells in the alveolar compartments. Cellular accumulation in bronchoalveolar lavage fluids and apoptosis of alveolar macrophages were significantly attenuated in mice infected with a mutant deficient in the expression of adenylate cyclase-hemolysin, indicating a role of this enzyme in these processes.

[Eosinophilia and interleukin-5: role in pulmonary atopic processes]. / L'eosinophile et l'interleukine-5: role dans les processus atopiques pulmonaires.

Infiltration of some tissues including the lung by activated eosinophil leukocytes is a characteristic feature of allergic disorders. In asthma patients, eosinophils have been shown to promote and prolong inflammation of bronchial mucosa. This effect is due to the release by eosinophils of numerous inflammation-stimulating mediators and cytokines as well as cationic proteins known to damage lung epithelium. Differentiation of eosinophils from their hematopoietic precursors as well as subsequent processes including maturation, chemotaxis, and activation are controlled mainly by interleukin-5 (IL-5). This cytokine is produced not only by a subpopulation of T-lymphocyte helpers, named Th2, but also by mastocytes and eosinophils themselves. A massive increase in IL-5 concentrations as well as in mRNA for IL-5 synthesis has been detected in serum, broncho-alveolar lavage fluid, and bronchial mucosa of asthmatic patients. In animal models mimicking clinical manifestations of lung allergy, neutralization of IL-5 using specific antibodies inhibits eosinophilic infiltration into the airways and improves respiratory function. These findings suggest that release of IL-5 during atopic disorders is a cardinal step in the development of inflammation and lesions in asthmatic patients. Treatment with drugs that selectively inhibit the synthesis and/or effects of IL-5 could represent a new therapeutic strategy not only for allergic disorders but also for other diseases associated with blood and tissue eosinophilia.

Role of eosinophilic airway inflammation in models of asthma

Eosinophils play a central role in the establishment and outcome of bronchial inflammation in asthma. Animal models of allergy are useful to answer questions related to mechanisms of allergic inflammation. We have used models of sensitized and boosted guinea pigs to investigate the nature of bronchial inflammation in allergic conditions. These animals develop marked bronchial infiltration composed mainly of CD4+ T-lymphocytes and eosinophils. Further provocation with antigen leads to degranulation of eosinophils and ulceration of the bronchial mucosa. Eosinophils are the first cells to increase in number in the mucosa after antigen challenge and depend on the expression of alpha4 integrin to adhere to the vascular endothelium and transmigrate to the mucosa. Blockage of alpha4 integrin expression with specific antibody prevent not only the transmigration of eosinophils but also the development of bronchial hyperresponsiveness (BHR) to agonists in sensitized and challenged animals, clearly suggesting a role for this cell type in this altered functional site. Moreover, introduction of antibody against Major Basic Protein into the airways also prevents the development of BHR in similar model. BHR can also be suppressed by the use of FK506, an immunosuppressor that reduces in almost 100 per cent the infiltration of eosinophils into the bronchi of allergic animals. These data support the concept that eosinophil is the most important pro-inflammatory factor in bronchial inflammation associated with allergy.