Evelyne Israël-Assayag

Effects of repeated swine building exposures on normal naive subjects

Exposure to swine confinement buildings has a negative impact on respiratory health. A short exposure to this environment results in an acute airway inflammatory response. The present study was performed to confirm and further define the acute effects of working in a swine building, and to determine whether these effects are reproducible. Seven previously nonexposed normal subjects underwent evaluations that included hourly measurement of forced expiratory volume in one second (FEV1), methacholine challenge (the provocative concentration producing a 20% fall in FEV1 (PC20)), bronchoalveolar lavage (BAL), nasal lavage (NL), and blood analyses, before (control) and after each of two 5 h exposures to a swine building environment. The exposures were conducted 8 days apart. The levels of total dust, endotoxins, and ammonia (NH3) in the confinement building were measured on each day of exposure. Both exposures resulted in a significant reduction in FEV1 (mean+/-SEM change in FEV1: control = 7+/-2%; exposure 1 = 15+/-3%; exposure 2 = 23+/-3%), decrease in PC20 (median value (25th-75th percentile): 223 (23-256), 20 (15-198) and 20 (11-71), respectively; p=0.05) and increase in BAL cells (129+/-20, 451+/-43 and 511+/-103x10(3) cells x mL(-1), respectively) and NL cells (6+/-4, 126+/-58 and 103+/-26x10(3) cells x mL(-1), respectively), mostly neutrophils. Levels of interleukin-8 (IL-8), but not interleukin-1 (IL-1) or tumour necrosis factors (TNF-alpha), increased both in BAL and nasal fluids with exposure. In normal naive subjects, repeated exposure to the environment of a swine building induced a marked and reproducible reduction in forced expiratory volume in one second, increase in airway responsiveness, and increased neutrophilic inflammatory response. These results could not be accounted for by any of the environmental factors measured.

Pathogenesis of hypersensitivity pneumonitis.

Purpose of reviewHypersensitivity pneumonitis is a group of immunologically mediated diseases caused by an abnormal response to a wide variety of inhaled antigens. Its pathogenesis is complex and involves many immunological concepts. This review discusses recent advances in our understanding of the pathogenesis of hypersensitivity pneumonitis. Recent findingsOver the last 3 years, several studies on the pathogenesis of hypersensitivity pneumonitis have been published. New antigens have been identified. We now have a better understanding of the role of inflammatory cells and mediators, and promoting and protective factors have been suggested. SummaryMost of the mechanisms involved in the pathogenesis of hypersensitivity pneumonitis remain incompletely understood. Current and future findings will not only help our understanding of the disease and its prevention, but also improve its treatment.

Modulation of eosinophil activation in vitro by a nicotinic receptor agonist

Nicotinic receptor agonists decreased the infiltration of eosinophils into the lung and airways in a mouse model of asthma. To better understand the mechanisms implicated in this anti‐inflammatory phenomenon, the expression of nicotinic acetylcholine receptors (nAChRs) and the effect of dimethylphenylpiperazinium (DMPP), a nonselective nAChR agonist, on human blood eosinophils were studied. The expression of α‐3, ‐4, and ‐7 nAChR subunits on human blood eosinophils was measured by cell ELISA and immunocytochemistry. mRNA expression for all three subunits was evaluated by quantitative RT‐PCR. The effect of DMPP on leukotriene C4 (LTC4) and matrix metalloproteinase‐9 (MMP‐9) production, eosinophil migration, and intracellular calcium mobilization was measured. The results show that the α‐3, ‐4, and ‐7 nAChR subunits and mRNAs are expressed by blood eosinophils. In vitro treatment of these cells with various concentrations of DMPP reduced platelet‐activating factor (PAF)‐induced LTC4 production significantly. DMPP (160 μM) decreased eotaxin, and 5‐oxo‐6,8,11,14‐eicosatetranoic acid induced eosinophil migration through Matrigel by 40.9% and 55.5%, respectively. This effect was reversed by the nAChR antagonist mecamylamine. In addition, DMPP reduced MMP‐9 release and the inositol 1,4,5‐triphosphate‐dependent intracellular calcium increase provoked by PAF. Taken together, these results indicate that functional nAChRs are expressed on eosinophils and that nAChR agonists down‐regulate eosinophil function in vitro. These anti‐inflammatory effects could be of interest in the treatment of allergic asthma.

Modulation of airway inflammation and resistance in mice by a nicotinic receptor agonist

Nicotinic agonists, including 1,1-dimethyl-4-phenylpiperazinium (DMPP), have anti-inflammatory properties and in some instances smooth muscle relaxing effects. Since inflammation and airway smooth muscle contraction are two major components of asthma, the present authors investigated the effects of DMPP on airway inflammation and airway resistance in a mouse model of asthma. Mice were sensitised and challenged with ovalbumin (OVA) and treated either intraperitoneally or intranasally with DMPP. The effect of DMPP was tested on airway inflammation, airway resistance and on the increase of intracellular calcium in bronchial smooth muscle cells. DMPP given either during sensitisation, OVA challenges or throughout the protocol prevented lung inflammation and decreased the serum level of OVA specific immunoglobulin E. DMPP administration reduced the number of total cells, lymphocytes and eosinophils in the bronchoalveolar lavage (BAL) fluid. Intranasal DMPP administration was as effective as dexamethasone (DEXA) in reducing total cell count and eosinophil counts in BAL fluid. DMPP, but not DEXA, reduced tissue inflammation. Intranasal DMPP, given 10 min before the test, reduced airway responsiveness to metacholine. DMPP also reduced the increase in intracellular calcium in response to bradykinin. In conclusion, these results show that 1,1-dimethyl-4-phenylpiperazinium reduces lung inflammation and prevents airway hyperresponsiveness in the mouse model of asthma.

Atrial natriuretic factor-induced cGMP accumulation in rat anterior pituitary cells in culture is not coupled to hormonal secretion.

While atrial natriuretic factor (ANF) does not influence ACTH secretion, it was reported to have a marked stimulatory effect on the intracellular accumulation of cGMP in rat anterior pituitary cells in culture. Since many biological actions of ANF appear coupled to its excitatory action on target cell guanylate cyclase, the current study was designed to characterize the ANF-induced cGMP response in anterior pituitary with a view to determining whether the nucleotide plays a regulatory role in the secretory function of this gland. A 3 min exposure of cells in primary culture to 300 nM ANF (99-126) or 100 microM sodium nitroprusside (SNP), a stimulator of guanylate cyclase, causes maximal 10- and 3-fold elevations of cGMP levels, respectively. Following a progressive decrease, 6- and 2-fold increases over basal cGMP levels are still observed after 180 min of incubation with ANF (99-126) and SNP, respectively. The half-maximal stimulation of cGMP accumulation induced by a 10 min exposure to ANF (99-126), or rat atriopeptin II (ANF 103-125) is observed at 9 +/- 2 and 125 +/- 22 nM, respectively. ANF fragments (99-109) and (111-126), as well as human cardiodilatin (hANF 1-16), do not alter cGMP levels. Basal and ANF-induced cGMP levels are at least 10-fold higher in cell populations enriched in gonadotrophs compared to gonadotroph-impoverished preparations. A 3 h incubation of cells with ANF (0.1-1000 nM), however, fails to modify spontaneous or LHRH-induced LH secretion. Similarly, ANF does not alter spontaneous release of GH, TSH or PRL. The data suggest indirectly that gonadotrophs represent a principal site at which ANF acts to stimulate cGMP synthesis, but that the nucleotide is not a specific regulator of the LH secretory process; nor is it generally involved as a second messenger in the secretory function of any cell type of the anterior pituitary gland.

Impaired function of regulatory T-cells in hypersensitivity pneumonitis

Hypersensitivity pneumonitis (HP) is characterised by lung lymphocytosis. Most individuals exposed to HP antigens remain asymptomatic. The mechanisms involved in the impaired immune tolerance leading to HP are unclear. Normally, T-regulatory (Treg)-cells control the immune response. The aim of the present study was to determine whether Treg-cell suppressive function deficiency can explain the uncontrolled inflammation in HP. Bronchoalveolar lavage (BAL) and blood samples were obtained from normal subjects, asymptomatic individuals and HP patients. BAL and blood Treg-cells were isolated. The ability of Treg-cells to suppress T-cell proliferation and the role of interleukin (IL)-17 was verified. BAL and blood Treg-cells from normal subjects suppressed the proliferative response of activated T-cells by 47.1 and 42%, respectively. BAL and blood Treg-cells from asymptomatic subjects had a slightly decreased activity and suppressed proliferation by 29.4 and 31.8%, respectively. BAL and blood Treg-cells from HP patients were totally nonfunctional and unable to suppress proliferation. Low levels of IL-17 were detected in sera and BAL from both normal and asymptomatic individuals, whereas measurable levels were found in patients. Treg-cells may be involved in antigen tolerance in asymptomatic subjects. Defective Treg-cell function, potentially caused by increased IL-17 production, could account for the exacerbated immune response characteristic of HP.

Altered immunosuppressive activity of alveolar macrophages in farmer's lung disease

Since normal alveolar macrophages (AMs) can suppress T-cell proliferation to mitogenic and antigenic stimuli both in vitro and in vivo, we questioned whether an altered AM immunosuppressive activity could account for the alveolar lymphocytosis observed in farmers lung (FL) and whether granulocyte/macrophage colony-stimulating factor (GM-CSF), a cytokine able to abrogate AM-induced immunosuppression, is involved in the process. The ability of different concentrations of AMs to inhibit lymphocyte proliferation in response to the T-cell-specific mitogen phytohaemagglutin (PHA) after in vitro culture was tested in three groups of subjects: 12 patients with FL; four asymptomatic farmers (AS); and six normal volunteers (N). Release of GM-CSF by AMs was also measured. At all ratios tested, AMs from patients with FL did not suppress the lymphoproliferation but instead had an enhancing effect. In AS, AMs enhanced the proliferation at a lower ratio but inhibited it at high ratios. In N subjects, as described previously, AMs increasingly inhibited the blastogenesis of lymphocytes (L) at increasing ratios of AM:L. In some patients with FL, AMs spontaneously released more GM-CSF than in normal volunteers (206 +/- 84 versus 29 +/- 14 pg.mL-1, respectively). In AS, GM-CSF release was intermediate (74 +/- 36 pg.mL-1). In conclusion, a defect in the ability of alveolar macrophages to suppress the proliferation of lymphocytes in the lung of patients with farmers lung is a major factor accounting for the development of the observed lymphocytic alveolitis. Granulocyte/macrophage colony-stimulating factor could be one factor which may contribute to this alteration.

Mature CD11c+ cells are enhanced in hypersensitivity pneumonitis

The present study verified the hypothesis that enhanced maturation of antigen-presenting CD11c+ cells could explain the viral-induced exacerbated immune response to Saccharopolyspora rectivirgula (SR), the main antigen responsible for farmer’s lung, a classic form of hypersensitivity pneumonitis (HP). Four groups of mice were studied: group 1 received intranasal instillations of saline; group 2 received instillations of SR for 12 weeks; group 3 received instillations of saline and a single infection with Sendai virus on week 3; and group 4 received instillations of SR for 12 weeks with a single administration of Sendai virus on week 3. On week 13, mice were sacrificed and bronchoalveolar lavage was performed. Lungs were harvested, digested with enzymes, and CD11c+ cells were analysed in flow cytometry with anti-CD11c, anti-CD86 and anti-major histocompatibility complex class II markers. Immunofluorescence studies were also performed with the same cell surface markers. Both flow cytometry and immunofluorescence results demonstrate that mature CD11c+ cells are significantly enhanced in SR-challenged mice simultaneously infected with Sendai virus, compared with other groups. These CD11c+ cells persist in the lung for 9 weeks after the virus infection. Maturation of CD11c+ cells could explain, at least in part, the virus-induced increased immune response to SR antigens in this model of HP, but mechanisms have still to be elucidated.

The role of viruses in the pathogenesis of hypersensitivity pneumonitis

Hypersensitivity pneumonitis (HP) is a worldwide lung disease caused by an immune response to a variety of mostly organic inhaled antigens. Only a small percentage (for example, 3 in 1000 dairy farmers) of subjects exposed to HP antigens develop the disease. It is possible that cofactors are needed to render the patient hypersensitive to environmental antigens; recent studies suggest that a viral infection could be such a trigger factor. Mice infected once with Sendai virus and simultaneously sensitized with HP antigens develop an enhanced response to the antigen that persists long after the transient viral infection has waned. Moreover, viral antigens were found in the lavage cells and lung tissue of patients with HP. Viral infections are known modulators of the immune response; viruses increase the antigen-presenting capacity of alveolar macrophages, decrease phagocytosis and antigen clearance, induce the release of pro-inflammatory cytokines, and favor the proliferation of Th1 T-lymphocytes (cells putatively associated with HP).

Role of interleukin-2 in the development and persistence of lymphocytic alveolitis in farmer's lung

Farmers lung (FL) is characterized by an intense lymphocytic alveolitis which persists after an acute episode with continuous exposure to the offending antigens. This study aimed to examine the role of interleukin-2 (IL-2) in the development and persistence of this lymphocytic alveolitis. Three groups of dairy farmers were studied: acute FL, ex-FL (past history of FL but no clinical evidence of active disease) and asymptomatic farmers (no lung disease). IL-2 was measured by enzyme immunosorbent assay and T-cell proliferation was evaluated by 3H-thymidine incorporation. Acute and ex-FL patients had more lymphocytes (p<0.01) and higher levels of IL-2 (p<0.05) in their bronchoalveolar lavage (BAL) than asymptomatic farmers. BAL T-lymphocytes from acute and ex-FL patients released considerable amounts of IL-2 after stimulation with concanavalin A and showed dose-dependent proliferative responses to IL-2. IL-2 production was decreased after treatment with prednisone. Acute FL patients, but not ex-FL, had higher levels of soluble CD25 in their serum than asymptomatics (p=0.009). These results suggest that interleukin-2 may play a role in farmers lung by providing a stimulus not only for the accumulation of lymphocytes but also for their persistence at the site of hypersensitivity reaction, and that the lung is a likely source of this cytokine in vivo.