Reassessing the role of internalin B in Listeria monocytogenes virulence using the epidemic strain F2365.

OBJECTIVES: To investigate the contribution to virulence of the surface protein internalin B (InlB) in the Listeria monocytogenes lineage I strain F2365, which caused a deadly listeriosis outbreak in California in 1985. METHODS: The F2365 strain displays a point mutation that hampers expression of InlB. We rescued the expression of InlB in the L. monocytogenes lineage I strain F2365 by introducing a point mutation in the codon 34 (TAA to CAA). We investigated its importance for bacterial virulence using in vitro cell infection systems and a murine intravenous infection model. RESULTS: In HeLa and JEG-3 cells, the F2365 InlB+ strain expressing InlB was ≈9-fold and ≈1.5-fold more invasive than F2365, respectively. In livers and spleens of infected mice at 72 hours after infection, bacterial counts for F2365 InlB+ were significantly higher compared to the F2365 strain (≈1 log more), and histopathologic assessment showed that the F2365 strain displayed a reduced number of necrotic foci compared to the F2365 InlB+ strain (Mann-Whitney test). CONCLUSIONS: InlB plays a critical role during infection of nonpregnant animals by a L. monocytogenes strain from lineage I. A spontaneous mutation in InlB could have prevented more severe human morbidity and mortality during the 1985 California listeriosis outbreak.

Elicitation of the allergic reaction in beta-lactoglobulin-sensitized Balb/c mice: biochemical and clinical manifestations differ according to the structure of the allergen used for challenge.

BACKGROUND: Mouse models of allergy are used to study the mechanisms of induction and perpetuation of bronchopulmonary hyper-reactivity (BHR) as related to eosinophils and specific IgE. OBJECTIVE: Our aim was to adapt the current model for the study of bovine beta-lactoglobulin (BLG), a major cow's milk allergen, and to further analyse the mechanisms of the acute and late allergic reaction. METHODS: Female Balb/c mice were sensitized intraperitoneally with BLG and the influence of the adjuvant and of the BLG dose on the IgE response was analysed, IgE and IgG1 epitopes being characterized. Once optimized, this model was applied to the study of the active phase of allergy in the respiratory tract after a single airway challenge using native or denatured BLG, which contains only linear epitopes. RESULTS: An immediate allergic reaction was characterized by the rapid release of histamine into the bronchoalveolar lavage fluids. Prostaglandin (PG)D2 was only present when the standard histamine-releasing agent compound 48/80 or denatured BLG were used as triggers, whereas native BLG induced leukotriene release. Twenty-four hours after challenge, BHR, eosinophil influx, IL-4 and IL-5 production, plasma exudation and mucus production were very much increased, differently depending on the allergen structure, and indicated the occurrence of the late allergic reaction. Our results show that the murine model can be used to study the mechanisms of allergy to clinically relevant antigens, such as those contained in cow's milk. The acute allergic reaction, which depends on the structural feature of the allergen, is composed of two distinct pathways characterized by peptido-leukotrienes or PGD2 production, which may result from distinct activation intensities of mast cells, leading to distinct late reactions. CONCLUSION: This study thus demonstrates a clear link between the structural feature of a protein, and the physiopathology of the experimental asthmatic reaction.

Persistence of bronchopulmonary hyper-reactivity and eosinophilic lung inflammation after anti-IL-5 or -IL-13 treatment in allergic BALB/c and IL-4Ralpha knockout mice.

BACKGROUND: Antigen-induced bronchopulmonary hyper-reactivity (BHR) is generally associated with eosinophilia. It involves cytokines produced by Th2 lymphocytes, including IL-4, IL-5 and IL-13, which are implicated in IgE production, eosinophil differentiation and attraction, and related events relevant to allergic inflammation, whose mechanisms remain unclear. OBJECTIVE: To investigate the mechanisms by which Th2 cytokines mediate eosinophilia and subsequent BHR using ovalbumin (OVA)-immunized and OVA-challenged IL-4Ralpha-/- and IL-4-/- mice, which fail to transduce and/or to produce IL-4 and IgE as compared with wild type (WT) mice, and specific neutralizing antibodies. METHODS: On days 0 and 7, mice were immunized subcutaneously (s.c.) with OVA. At day 14, anti-IL-5 or anti-IL-13 antibodies were administered intranasally and/or intravenously before allergenic challenge. Different functional and cellular parameters were studied in vivo and cytokine production was followed with a newly described ex vivo procedure using lung explants. RESULTS: IL-4Ralpha-/- and IL-4-/- mice developed BHR and pulmonary eosinophilia, even though eosinophil recruitment to the bronchoalveolar liquid lavage (BALF) was reduced. In vivo, IL-4-/- and IL-4Ralpha-/- mice produced, respectively, no or reduced amounts of IL-5 in the BALF/serum as compared with WT mice, whereas no IL-13 in the BALF was detected. By contrast, ex vivo, surviving lung explants from WT and IL-4-/- or IL-4Ralpha-/- mice produced IL-13 and large amounts of IL-5. The neutralization of IL-5 in vivo (BALF and serum) and ex vivo (from lung explant) in IL-4Ralpha-/- and WT mice failed to suppress BHR and lung eosinophilia, and to modify IL-13 production ex vivo. In addition, neutralization of IL-13 in vivo from lung explant also failed to abrogate BHR and lung eosinophilia, whereas IL-5 was unchanged. CONCLUSION: Antigen-induced BHR can develop independently from IL-4, IL-5 or IL-13 and from the IL-4alpha receptor chain, suggesting a possible novel IL-4, IL-5 and IL-13-independent pathway for the development of BHR in allergic BALB/c mice. The failure of IL-5 or IL-13 antibodies to prevent BHR in IL-4Ralpha-/- mice suggests that neither is indispensable for BHR but does not exclude a role for lung tissue eosinophilia.

Development of mixed Th1/Th2 type immune response and protection against Mycobacterium tuberculosis after rectal or subcutaneous immunization of newborn and adult mice with Mycobacterium bovis BCG.

Cell-mediated immunity plays a key role in containing the growth of Mycobacterium tuberculosis in the host. The induction of an antibody response or a mixed cell-mediated and humoral response is frequently associated with tuberculosis disease or a decrease in the ability to control M. tuberculosis load. We recently reported the induction of similar immune responses and protection by rectal, subcutaneous (SC) or intradermal administration of Mycobacterium bovis BCG in adult mice, guinea pigs and macaques. The rectal immunization, which did not induce the side-effects associated with parenteral routes (axillary adenitis) and which could be used to reduce the risks of viral transmission associated with unsafe injections in the developing world, was analysed and compared in newborn and adult BALB/c mice. The rectal and SC immunization induced, in mice immunized as newborns or as adults, a mixed T helper 1/T helper 2 (Th1/Th2) immune response; however, particularly in adult mice, after SC administration of BCG, the level of Th2 immune response is significantly higher than it is by the rectal route. Six months after immunization with BCG, rectal and SC delivery induced similar levels of protective immunity against a virulent challenge with M. tuberculosis strain (H37Rv) in mice immunized as adults, but the rectal BCG delivery triggered stronger protection than the SC delivery if mice were immunized as newborns.

Suppression of asthma-like responses in different mouse strains by oral tolerance.

In this study we examined the effect of oral antigen (Ag) administration on the development of experimental asthma in different mouse strains. We selected BALB/c, BP2, CBA/Ca interleukin (IL)-5 transgenic, and BALB/c T-cell receptor-delta-deficient mouse strains because they exhibit different aspects of the asthma syndrome. Mice exposed to 1% ovalbumin (OVA), dissolved in the drinking water for 5 consecutive days, became unresponsive to subsequent immunogenic OVA challenges. This regimen of OVA administration induced Ag-specific unresponsiveness in all mouse strains tested, including gammadelta-deficient mice that are said to be resistant to tolerance induction. The Ag-specific unresponsiveness was characterized by reduced (almost absent) airway eosinophilic inflammation, airway hyperreactivity, and mucus production; also by low levels of T helper (Th) 2-type cytokines in bronchoalveolar lavage fluid, and decreased immunoglobulin (Ig) G1 and IgE OVA-specific antibody production. The unresponsive state was not associated with increased levels of the suppressive cytokines IL-10 and transforming growth factor (TGF)-beta or with immune deviation toward the Th1 pathway due to increased levels of interferon-gamma and IL-12. Moreover, treatment with anti- TGF-beta antibodies did not abrogate oral tolerance. Oral Ag administration was quite effective in suppressing the development of key features of asthma when initiated after primary immunization (Day 0) or after booster (Day 7), but not after challenge (Day 14) when it increased allergic responses. Collectively, our findings show for the first time the beneficial and detrimental effects of oral Ag administration on the development of experimental asthma.

Effects of Mycobacterium bovis BCG on the development of allergic inflammation and bronchial hyperresponsiveness in hyper-IgE BP2 mice vaccinated as newborns.

Asthma may result from excessive Th-2 response in children not previously exposed to Th-1-inducing infections. We tested the hypothesis that BCG vaccination in Th-2-susceptible newborn BP2 mice blocks allergic inflammation and bronchial hyperreactivity (BHR). Ten day-old BP2 mice received 10(5) CFU of BCG 1173P2 intranasally (IN), and 6, 10 or 14 weeks thereafter were sensitized with 100 microg ovalbumin (OVA) in aluminium hydroxide twice subcutaneously (SC) at 1 week interval, and challenged 1 week after the second sensitization with 10 microg OVA IN. Compared to OVA-challenged unvaccinated mice, those that received BCG 8 weeks before challenge developed intense bronchial inflammation, BHR, and high IgE titers. Inflammation involved T cells, macrophages, dendritic cells and was accompanied by increased levels of Interleukin-5 (IL-5) in the bronchoalveolar lavages (BAL). However, animals challenged 16 weeks after BCG vaccination did not develop BHR nor bronchial hypereosinophilia, and showed reduced IgE levels. Bronchial infiltration by immunocompetent cells was also significantly reduced. Increased levels of gamma-interferon (IFN-gamma) after in vitro stimulation of tracheo-bronchial lymph node cells accompanied this blockage, but levels of IL-5 remained high. We demonstrate that 16 weeks after vaccination with BCG in newborn BP2 mice which have a high Th-2 background, allergic inflammation and BHR were blocked, even though a clear Th-1 shift was not achieved.

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.

Systemic administration of endotoxin induces bronchopulmonary hyperreactivity dissociated from TNF-alpha formation and neutrophil sequestration into the murine lungs.

Bronchopulmonary hyperreactivity (BHR), an increased responsiveness to nonspecific bronchoconstrictor agents, is a well-known characteristic of bronchial asthma. It has been recently suggested that the severity of this disease is related to the endotoxin content of house dust. In the present report, it is shown that the i.p. administration of bacterial LPS to mice is followed by a marked early dose-dependent BHR in response to methacholine. The microscopic examination showed no ultrastructural lesions of the lungs or of the airways, but a marked neutrophil accumulation in the capillaries, as confirmed by an increase of the lung content in the neutrophil enzyme marker myeloperoxidase. In parallel, high levels of TNF-alpha were found in plasma as well as its transcripts in the lung tissues. Using immunologic (anti-TNF-alpha and anti-granulocyte Abs), and pharmacologic (dexamethasone and vinblastine) tools, it is demonstrated that BHR is apparently neither related to the presence of neutrophils in the pulmonary microvasculature nor to the synthesis of TNF-alpha.

The LPS-induced neutrophil recruitment into rat air pouches is mediated by TNFalpha: likely macrophage origin.

The role of resident cells during the lipopolysaccharide (LPS)-induced neutrophil recruitment into rat air pouches was investigated. In this model, LPS (Escherichia coli, O55: B5 strain; 2-2000 ng) induced a dose- and time-dependent neutrophil recruitment accompanied by the generation of a tumour necrosis factor-alpha (TNFalpha)-like activity. Dexamethasone (0.05-5 mug) and cycloheximide (6 ng), injected 2 h before LPS into the pouches, inhibited the neutrophil recruitment and the generation of the TNFalpha-like activity, while the H1-receptor antagonist mepyramine (1 and 4 mg/kg, i.p., 0.5 h before LPS) and the PAF-receptor antagonist WEB 2170 (0.05 and 1 mg/kg, i.p., 0.5 h before LPS) had no effect. Purified alveolar macrophages (AM) were used to replenish the pouches of cycloheximide-treated recipient rats. AM provided by PBS-treated animals led to the recovery of the LPS-induced neutrophil recruitment and of the TNFalpha-like formation contrasting with those from cycloheximide-treated animals (1 mg/kg, i.p.). When delivered in situ, liposome-encapsulated clodronate, a macrophage depletor, significantly impaired both the LPSinduced neutrophil recruitment and the TNFalpha-like activity. An anti-murine TNFalpha polyclonal antibody (0.5 h before LPS) was also effective. These results emphasize the pivotal role of macrophages for LPS-induced neutrophil recruitment via the formation of TNFalpha.

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.

In vivo neutralization of eosinophil-derived major basic protein inhibits antigen-induced bronchial hyperreactivity in sensitized guinea pigs.

This study examines the effect of purified rabbit antiguinea pig eosinophil-derived major basic protein (MBP) Ig on antigen-induced bronchial hyperreactivity to inhaled acetylcholine in aerosol-sensitized guinea pigs. Ovalbumin inhalation by sensitized guinea pigs induced a rise in the numbers of eosinophils and in the levels of MBP in the bronchoalveolar lavage fluid, which peaked at 24 h and resolved at 72 h. Antigen-challenged animals exhibited bronchial hyperreactivity to inhale acetylcholine at 72 h, but not at 6 or 24 h. The intranasal administration of 200 microliter of purified rabbit anti-guinea pig MBP Ig, at 2.5 mg/ml, but not of the control preimmune rabbit Ig, 1 h before and 5 h after ovalbumin inhalation suppressed bronchial hyperreactivity to acetylcholine at 72 h without affecting the number of eosinophils accumulating in the bronchoalveolar lavage fluid. These findings indicate that antigen challenge in sensitized guinea pigs is followed by early eosinophil infiltration and activation within the airways and by late bronchial hyperreactivity. Neutralization of endogenously secreted MBP by a specific antiserum prevented antigen-induced bronchial hyperreactivity, suggesting that eosinophil degranulation plays an important role in the alterations of bronchopulmonary function in the guinea pig.

IL-5 drives eosinophils from bone marrow to blood and tissues in a guinea-pig model of visceral larva migrans syndrome.

This study was undertaken to evaluate the role of IL-5 in eosinophil migration and in the maintenance of eosinophilia in a guinea-pig model of visceral larva migrans syndrome. The results show that the infection of animals with Toxocara canis induced an early increase in serum IL-5 levels that might be essential for eosinophil differentiation and proliferation and for the development of eosinophilia. When infected guinea-pigs were treated with mAb anti-IL-5 (TRFK-5) given at the same time or 1 or 3 days after infection, there was a high percentage of reduction of eosinophil counts 18 days after infection. However, when the mAb was administered during the peak of eosinophilia, there was high inhibition in blood, no inhibition in bronchoalveolar lavage fluid (BALF) or peritoneum and an increase in eosinophil numbers in bone marrow. Thus, a basic level of IL-5 may be essential to drive eosinophils from bone marrow to blood and tissues, and for the maintenance of eosinophilia in infected animals. We may also conclude that when eosinophils have already migrated to the lungs, TRFK-5 has no power to inhibit eosinophilia, which is also under control of local lung cells producing IL-5. In this way, only one later TRFK-5 treatment may not be sufficient to modify the lung parenchyma microenvironment, since T. canis antigens had already stimulated some cell populations to produce IL-5.

Fenspiride: an anti-inflammatory drug with potential benefits in the treatment of endotoxemia.

Using a model of endotoxemia triggered by the intravenous injection of bacterial lipopolysaccharide (0.1 and 1 mg/kg) to guinea-pigs, we investigated the interference of fenspiride, an anti-inflammatory drug recommended for the treatment of inflammatory diseases of the upper respiratory tract. Administered orally at 60 mg/kg, fenspiride reduced the lipopolysaccharide-induced early rise of tumor necrosis factor concentrations in serum (4.2 +/- 0.9 vs. 2.3 +/- 0.5 ng/ml, P < 0.05) and in the bronchoalveolar lavage fluid (55.7 +/- 20 vs. 19.7 +/- 7.5 ng/ml, P < 0.05). The lipopolysaccharide-induced primed stimulation of alveolar macrophages, defined as their enhanced release of arachidonic acid metabolites as compared to cells from untreated controls upon stimulation with N-formyl-methionyl-phenylalanine was also reduced by fenspiride (1551.5 +/- 183.7 vs. 771.5 +/- 237.5 pg/mu g protein, P < 0.05 for thromboxane B2 and 12.6 +/- 4.9 vs. 3.6 +/- 0.9 pg/ mu g protein, P < 0.05 for leukotriene C4). Finally, fenspiride reduced the increased serum concentrations of extracellular type II phospholipase A2 (3.9 +/- 1.2 vs. 1.2 +/- 0.1 nmol/ml per min, P < 0.01), the intensity of the neutrophilic alveolar invasion and the lethality due to the lipopolysaccharide. The protective effect of fenspiride may result from the inhibition of the formation of tumor necrosis factor, a major mediator of the effects of lipopolysaccharide.

Modulation by dexamethasone of phospholipase A2 activities in endotoxemic guinea pigs.

One hour after lipopolysaccharide (LPS) administration (intravenous) in guinea pigs, alveolar macrophages are primed for an ex vivo increased secretion of arachidonic acid metabolites from the cyclooxygenase and the lipoxygenase pathways, with challenge by a second stimulus. At the same time, maximal levels of tumor necrosis factor-alpha (TNF-alpha) are observed in the circulation and in the bronchoalveolar lavage fluid. An extracellular form of phospholipase A2, corresponding probably to the low-molecular-mass type II enzyme, known to accumulate in inflammatory exudates, appears later in the serum of guinea pigs, to reach maximal levels 6 h after the LPS. Unlike the intracellular enzyme, extracellular phospholipase A2 is not increased by LPS in alveolar macrophages or in bronchoalveolar lavage fluids. After 24 h, at the time when neither TNF-alpha nor extracellular phospholipase A2 is present and priming of macrophages is over, maximal neutrophil infiltration is observed in the alveolar space of LPS-treated guinea pigs. Dexamethasone administered repeatedly during 3 days (subcutaneous) before the LPS challenge prevented both early events such as the macrophage priming and the TNF-alpha appearance and later events such as extracellular phospholipase A2 release and neutrophil recruitment.

Interleukin-5 and eosinophil cationic protein in nasal lavages of rhinitis patients.

The production of interleukin-5 and eosinophil cationic protein (ECP) in the nasal cavity was examined in 24 patients with rhinitis who were allergic to the house dust mite. During a double-blind placebo-controlled cross-over study, fluticasone propionate aqueous nasal spray (200 micrograms) was administered twice daily for 2 weeks. After four basal nasal lavages provocation with house dust mite extract was performed and nasal lavages were collected every hour for 9.5 h. Interleukin-5 was present in detectable amounts in nasal lavages from patients allergic to house dust mite. Nasal challenge with house dust mite extract caused immediate nasal symptoms and increased levels of interleukin-5. Between 3.5 and 8.5 h after the challenge symptoms recurred and interleukin-5 levels increased, reflecting a late phase reaction. Eosinophil cationic protein, a marker of activated eosinophils, was released between 6.5 and 9.5 h after challenge. Treatment with fluticasone propionate (as an aqueous nasal spray) significantly decreased the evoked interleukin-5 and ECP levels in the late phase reaction. This response was correlated with an improved symptom score. This could indicate that the number and activity of eosinophils are increased during the late phase allergic reaction, a response that is inhibited by corticosteroids.

Binding of serum autoantibodies to sialidase-treated tracheal epithelial cells. Determination of autoantibodies isotypes in normal and influenza virus infected guinea pig sera.

Cultured epithelial cells isolated from guinea pig trachea were treated with Vibrio cholerae sialidase. The treatment was not cytotoxic and resulted in membrane desialylation as assessed by measurement of sialic acids released, along with an increased fixation of the galactose-specific lectin peanut agglutinin. After incubation in serum from normal guinea pigs, membrane-bound immunoglobulins were detected using peroxidase-labelled antibodies. Sialidase-treated cells bound significantly more IgM than controls (P < 0.0005), whereas binding of IgG was not significantly different between treated and untreated cells (0.1 < P < 0.375); IgA were never detected. In influenza-infected guinea-pigs, as assessed by reactivity with peanut agglutinin, the tracheal and lung epithelium, as well as alveolar cells were hyposialylated. In these animals, the level of serum IgG autoantibodies capable to bind sialidase treated cultured cells increased, while the level of IgM autoantibodies did not change. These autoantibodies may participate in cellular dysfunctions and modified bronchoreactivity that occur during infection of the respiratory tract by sialidase-producing microorganisms, either through activation of the complement system, or subsequently to their reaction with cells expressing membrane complement and/or Fc receptors.

Activation and damage of cultured airway epithelial cells by human elastase and cathepsin G.

Accumulation of polymorphonuclear neutrophils (PMN) and epithelium damage have often been described during airway inflammation. We studied the effects of two PMN-derived proteinases, namely elastase and cathepsin G, on guinea-pig tracheal epithelial cells in culture. Both proteinases activated tracheal epithelial cells in terms of prostaglandin (PG) E2 production. A concentration- and time-dependent effect was observed with 10 micrograms/ml and 6 h as the optimal conditions for both enzymes. Optical microscopic studies confirmed an effect on tracheal epithelial cells as intercellular gaps were observed upon incubation of the monolayers with proteinases. A small cytotoxic effect was observed after 1 h incubation but remained stable up to 6 h. This cytotoxic effect, more pronounced with elastase than with cathepsin G, was dissociated from PGE2 formation.

Guinea pig tracheal epithelial cells in primary culture: morphological and metabolic characterization.

The tracheo-bronchial epithelial cells are the first cells of the respiratory tract to encounter inhaled pathogens or allergens. In order to study these interactions in vitro we developed a culture of epithelial cells from guinea pig trachea which were kept as a primary culture for 3-13 days. Electron microscopy showed that the specific ultrastructure of epithelial cells was maintained, particularly cytokeratin intermediate filaments detected by immunofluorescence. The cells in culture produced eicosanoid metabolites, mainly leukotriene B4 and prostaglandin E2 spontaneously and upon calcium ionophore A23187 stimulation.