Mass or total surface area with aerosol size distribution as exposure metrics for inflammatory, cytotoxic and oxidative lung responses in rats exposed to titanium dioxide nanoparticles.

There is currently no consensus on the best exposure metric(s) for expressing nanoparticle (NP) dose. Although surface area has been extensively studied for inflammatory responses, it has not been as thoroughly validated for cytotoxicity or oxidative stress effects. Since inhaled NPs deposit and interact with lung cells based on agglomerate size, we hypothesize that mass concentration combined with aerosol size distribution is suitable for NP risk assessment. The objective of this study was to evaluate different exposure metrics for inhaled 5 nm titanium dioxide aerosols composed of small (SA < 100 nm) or large (LA > 100 nm) agglomerates at 2, 7, and 20 mg/m3 on rat lung inflammatory, cytotoxicity, and oxidative stress responses. We found a significant positive correlation ( r = 0.98, p < 0.01) with the inflammatory reaction, measured by the number of neutrophils and the mass concentration when considering all six (SA + LA) aerosols. This correlation was similar ( r = 0.87) for total surface area. Regarding cytotoxicity and oxidative stress responses, measured by lactate dehydrogenase and 8-isoprostane, respectively, and mass or total surface area as an exposure metric, we observed significant positive correlations only with SA aerosols for both the mass concentration and size distribution ( r > 0.91, p < 0.01), as well as for the total surface area ( r > 0.97, p < 0.01). These data show that mass or total surface area concentrations alone are insufficient to adequately predict oxidant and cytotoxic pulmonary effects. Overall, our study indicates that considering NP size distribution along with mass or total surface area concentrations contributes to a more mechanistic discrimination of pulmonary responses to NP exposure.

Protein Corona Formation on Magnetite Nanoparticles: Effects of Culture Medium Composition, and Its Consequences on Superparamagnetic Nanoparticle Cytotoxicity.

The physicochemical properties and potential cytotoxicity of nanoparticles (NPs) are significantly influenced by their inter- action with proteins, which results in corona formation. Here, we have determined whether corona formation, resulting from interactions between superparamagnetic iron oxide nanoparticles (SPIONs) and different cell culture media, may have consequences for driving NP toxic effects. To address this issue, complementary methods were used. The deter- mination of the hydrodynamic size distribution by ζ (zeta) potential measurement indicated that SPIONs were negatively charged under all conditions but that the actual charge was differed with the cell culture medium used. In vitro protein adsorption studies were carried out using the Bradford protein assay and Fourier transform infrared spectroscopy (FTIR). The Bradford assay revealed that the concentration of unadsorbed proteins and other biomolecules decreased when the SPION concentration increased. FTIR showed that the proteins were, indeed, adsorbed onto the NP surface. This was followed by matrix-assisted laser desorption/ionization time-of-flight secondary ion mass spectrometry (MALDI TOF-SIMS), to identify the adsorbed proteins. Ultimately, three different cell viability assays led to the conclusion that the SPIONs were not toxic for all the concentrations used here. In summary, we found that corona formation on the SPIONs depends on the composition of the culture media but has no consequence for nanotoxicity. We have shown that the application of complementary methods has provided novel insights into SPION/protein interactions.

Effects of inhaled nano-TiO2 aerosols showing two distinct agglomeration states on rat lungs.

Nano-aerosols composed of large agglomerates (LA) (>100nm) are more likely to promote pulmonary clearance via macrophages phagocytosis. Small agglomerates (SA) (<100nm) seem to escape this first defense mechanism and are more likely to interact directly with biological material. These different mechanisms can influence pulmonary toxicity. This hypothesis was evaluated by comparing the relative pulmonary toxicity induced by aerosolized nano-TiO(2) showing two different agglomeration states: SA (<100nm) and LA (>100nm) at mass concentrations of 2 or 7mg/m(3). Groups of Fisher 344 male rats were nose-only exposed for 6h. The median number aerodynamic diameters were 30 and 185nm at 2mg/m(3), and 31 and 194nm at 7mg/m(3). We found in rat's bronchoalveolar lavage fluids (BALF) a significant 2.1-fold increase in the number of neutrophils (p<0.05) in the group exposed to the 7mg/m(3) LA nano-aerosol suggesting a mild inflammatory response. Rats exposed to the 7mg/m(3) SA nano-aerosol showed a 1.8-fold increase in LDH activity and 8-isoprostane concentration in BALF, providing evidence for cytotoxic and oxidative stress effects. Our results indicate that biological responses to nanoparticles (NP) might depend on the dimension and concentration of NP agglomerates.

Association of bronchial reactivity to occupational agents with methacholine reactivity, sputum cells and immunoglobulin E-mediated reactivity.

BACKGROUND: Bronchial responsiveness and IgE-mediated reactivity are associated with specific bronchial reactivity to allergens. OBJECTIVE: Our aim was to examine whether airway inflammation also plays a role. METHODS: Retrospective analysis of all subjects who underwent specific inhalation challenges in the investigation of occupational asthma (OA) since 2000. Responsiveness to methacholine (PC(20) ) and levels of eosinophils and neutrophils in induced sputum on the control day were associated with the presence of OA (positive-specific inhalation challenge). In a sample of subjects exposed to wheat flour, we also examined the role of specific IgE- mediated reactivity (skin reactivity, specific IgE). RESULTS: PC(20) level was significantly more often normal in subjects with OA (35 of 129, 27% instances) by comparison with non-OA (15 of 189, 8% instances), but the positive predictive value of responsiveness to methacholine for OA was low (35%). Coupling information on the level of eosinophils to responsiveness to methacholine increased positive predictive values for OA from 39% to 69% depending on the thresholds used. The best balance of positive (69%) and negative (60%) predictive values was obtained in the case of normal PC(20) and eosinophils ≥3%. In a multivariate analysis carried out in 34 subjects exposed to wheat flour, responsiveness to methacholine, sputum eosinophils, skin weal size and levels of specific IgE were all significantly associated with OA to wheat flour. CONCLUSION AND CLINICAL RELEVANCE: Information on the level of sputum eosinophils in addition to PC(20) provides a better association with OA vs. non-OA when PC(20) is normal. Levels of sputum eosinophils in addition to PC(20) and IgE-mediated reactivity increase the likelihood of OA due to wheat flour.

Matrix metalloproteinases, IL-8 and glutathione in the prognosis of workers exposed to chlorine.

BACKGROUND: Workers exposed to chlorine may be at risk of deterioration in FEV1. METHODS: A prospective study of 72 workers examined over a 5.8 +/- 1.9 year period. A sample of induced sputum for cells and mediators was obtained in 69 subjects at baseline (Vb) and in 36 both at Vb and at follow-up (Vf). RESULTS: Sixty-four workers (89%) experienced at least one accidental inhalation of chlorine in the interval. The mean decrease in FEV1 was 30 ml/year and thus was within normal limits. Among the analysed remodelling markers, the level of the MMP-9-TIMP-1 complex, but not of free MMP-9 and TIMP-1, significantly diminished from Vb to Vf. We found significant correlations between neutrophils, IL-8, MMP-9 and MMP9-TIMP-1 complex at Vb and Vf. While levels of total glutathione, IL-8, MMP9, TIMP-1 and MMP9-TIMP-1 complex were highly correlated with each other at Vb, this was inconstant at Vf. Levels of MMP9-TIMP1 complex and of TIMP1 at Vf were significantly lower in workers reporting chlorine puffs with mild acute respiratory symptoms between visits compared to those who had no, or asymptomatic inhalations (P = 0.03 and 0.02, respectively). The fall in FEV1 from Vb to Vf was significantly correlated with levels of glutathione at Vb. Cough between visits was associated with a decrease in FEV1 (P = 0.06). CONCLUSION: Although no accelerated loss in FEV1 was documented in these workers exposed to chlorine, subjects with a greater fall in FEV1 were more likely to report cough and have higher levels of total glutathione at Vb.

Conjugates of ovalbumin and monomethoxypolyethylene glycol abolish late allergic responses and decrease IL-4 and IL-5 mRNA expression in the rat.

The purpose of this study was to test the therapeutic potential of monomethoxypolyethylene glycol (mPEG) conjugated-allergen using a rodent model of allergic asthma. Previously, this conjugate has been shown to possess the dual capacity of inducing long-term ovalbumin (OA)-specific suppression of the antibody response and inactivating rat mast cells that have been sensitized with murine IgE to OA. Ovalbumin sensitized and challenged Brown Norway rats were studied. Fourteen days after sensitization, a test group of six rats received mPEG-OA solution intratracheally and were challenged 30 min later with aerosolized OA. Another group of seven sensitized rats was similarly challenged with OA 30 min after intratracheal administration of normal saline. A group of six sensitized rats received mPEG-OA solution intratracheally but were challenged with normal saline. Another group of seven sensitized rats received mPEG-BSA solution intratracheally and were challenged 30 min later with aerosolized OA. A final group of five unsensitized rats were neither challenged nor medicated intratracheally. Pulmonary resistance was measured before and for 8 h following inhalation challenge. mPEG-OA treatment had an inhibitory effect on the allergic late airway response, but the early response was not significantly altered. Both mPEG-OA and mPEG-BSA reduced the total cells, eosinophils and neutrophils, in bronchoalveolar lavage and decreased the expression of IL-4, IL-5 and IFN-gamma mRNA. In conclusion, mPEG-OA can prevent the development of allergen-induced late airway responses and reduce airway Th2-type cytokine expression whereas mPEG conjugated to an irrelevant antigen (BSA) is anti-inflammatory but does not affect the late response.

Neutrophilic airway inflammation in horses with heaves is characterized by a Th2-type cytokine profile.

Heaves in horses shares many similarities with human asthma, including lower airway inflammation, reversible airway obstruction, and bronchial hyperresponsiveness. Extrinsic asthma is an allergic response to environmental allergens and a similar immunologic mechanism may be implicated in heaves. It is now recognized that a Th2 subset of CD4+ lymphocytes is associated with allergic diseases such as atopic asthma. The purpose of this study was to determine whether airway inflammation in heaves is associated with a pattern of expression of cytokine suggestive of a Th2 type response. The expression of mRNA, encoding interleukin (IL)-4, IL-5, and interferon gamma (IFN-gamma) was measured in bronchoalveolar cells from seven horses with heaves and five control horses, using in situ hybridization and radiolabeled equine-specific cRNA probes coding for these cytokines. Bronchoalveolar cells of horses with heaves had an increased expression of IL-4 (p = 0.01) and IL-5 (p = 0.02) mRNA and a decreased expression of INF-gamma (p = 0.01) compared with control horses. Here we show that inflammatory cells in lungs from horses with heaves display a Th2-type cytokine profile that is consistent with the hypothesis that heaves is an allergic condition with similarity to human asthma.

Characterization of airway inflammation after repeated exposures to occupational agents.

BACKGROUND: Little is known about the comparative kinetics of eosinophil recruitment after exposure to low- and high-molecular-weight sensitizers in subjects with occupational asthma (OA). OBJECTIVES: The aims of the study were to investigate the kinetics of changes in inflammatory mediators associated with eosinophil infiltration (IL-5 and eotaxin) and to examine the nature of the airway inflammation induced in response to different types of occupational agents. METHODS: We investigated 15 subjects with OA caused by high- and low-molecular-weight agents. The subjects were exposed to increasing doses of the relevant occupational agent over 3 to 4 days until a 20% fall in FEV(1) occurred. Methacholine challenge and sputum induction were performed at the end of each day of exposure. Sputum samples were assessed for differential cell counts, including eosinophils, IL-5, and eotaxin messenger RNA. RESULTS: There was an increase in sputum eosinophils, eotaxin, and IL-5 on the day preceding the occurrence of asthmatic reaction, although there was no change in functional parameters (FEV(1) and PC(20)). Increase in sputum eosinophils was more prominent in subjects exposed to low-molecular-weight agents than to high-molecular-weight agents. CONCLUSION: Changes in eosinophils, IL-5, and eotaxin precede functional changes after exposure to occupational agents in subjects with OA. Eosinophil inflammation is a feature of exposure to both high- and low-molecular-weight agents. Induced sputum may be a useful tool in the early diagnosis of OA.

Therapeutic interventions aimed at reducing airway smooth muscle growth.

The airways of asthmatic subjects undergo complex changes which affect all compartments of the airway wall. The airway smooth muscle (ASM) is particularly affected and is increased in mass. Both hyperplasia and hypertrophy probably contribute to the altered mass of muscle which is a potential cause of airway hyperresponsiveness. The impedance to smooth muscle shortening resulting from the constitutive properties of the airway wall and the elasticity of the parenchyma may be more easily overcome by the excess muscle of the asthmatic airway. The ASM may also undergo important changes in its functional characteristics as a result of allergic sensitization and challenge. Increases in maximal shortening velocity, phospholipase C activity and membrane potential are some of the changes in the characteristics of sensitized ASM. The ASM may also change its phenotype from a contractile to a secretory tissue in response to the stimulus of airway inflammation and in doing so may contribute to the inflammatory process itself. The current therapies for asthma have the potential to counter some of the adverse effects of airway modelling, in particular in so far as the ASM is concerned. These effects should be kept in mind when considering the rationale for effective maintenance therapy for asthma.

Dichotomy between neurokinin receptor actions in modulating allergic airway responses in an animal model of helper T cell type 2 cytokine-associated inflammation.

Neurokinins (NKs), which include substance P (SP) and neurokinin A (NKA), act through NK-1 and NK-2 receptors. There is considerable evidence of interaction between the neurogenic and the immune systems, and NKs are candidates for mediating such interactions. We hypothesized that selective inhibition of pulmonary NK-1 or NK-2 receptors may modulate immune responses so as to prevent the development of allergic airway responses in the atopic BN rat sensitized to ovalbumin (OA). To address this hypothesis, we have validated our animal model by showing that NK-1 and NK-2 receptors are expressed in the lungs, and that SP is released in the airways after allergen challenge. The selective NK-1 (CP-99,994) or NK-2 (SR-48968) antagonists before allergen challenge failed to reduce the allergic early airway responses. In contrast, both neurokinin antagonists decreased allergen-induced late airway responses in OA-challenged animals. However, only the NK-2 antagonist decreased the eosinophil numbers in the bronchoalveolar lavage (BAL). Likewise, the NK-2, but not NK-1, antagonist decreased both Th1 (INF-gamma) and Th2 (IL-4 and -5) cytokine expression in BAL cells by in situ hybridization. These results provide initial in vivo evidence linking neurokinins to the regulation of cytokine expression in cells without discrimination as to their phenotype. We conclude that there is a dichotomy between NK receptors in the modulation of the allergic airway inflammation, which has important implications for future therapeutic strategies for asthma using the NK antagonists.

Role of cysteinyl leukotrienes in CD4(+) T cell-driven late allergic airway responses.

Cysteinyl leukotrienes (cys LTs) play an important role in late responses to allergen challenge of actively sensitized rats. The aim of this study was to determine whether T cell-dependent late airway responses (LARs) also are mediated by cys LTs. To do this we tested the effects of the selective and potent LTD(4) antagonist pranlukast on airway responses to ovalbumin (OVA) challenge of naive recipients of CD4(+) T cells isolated from the cervical lymph nodes of OVA-sensitized donor rats. CD4(+) T cells (5 million) were purified by immunomagnetic separation and administered i.p. 2 days before OVA challenge. The pulmonary resistance was measured for 8 h after challenge and bronchoalveolar lavage (BAL) was performed for analysis of leukocytes and major basic protein-positive cells. The LAR, determined as the area under the curve of pulmonary resistance against time from 3 to 8 h after challenge, was 8.9 +/- 1.79 cm H(2)O/ml/s x min after OVA compared with 2.8 +/- 0.50 cm H(2)O/ml/s x min (P <.01) after OVA and pranlukast treatment. The total cell count in BAL was not significantly greater in the OVA challenged group (3.55 +/- 0.41 x 10(6) cells) compared with the OVA- and pranlukast-treated group (2.65 +/- 0.45 x 10(6) cells). However, lymphocytes and eosinophils were reduced in numbers by pranlukast. Interleukin-5 mRNA-positive cells were diminished by 50% in pranlukast-treated animals. In conclusion, pranlukast inhibits LAR, BAL eosinophilia, and Interleukin-5 expression in rats with adoptively transferred LAR, indicating an important role for cys LTs in these T cell-driven responses.

Evidence for the activation of blood complement in Sephadex beads-induced lung inflammation in guinea pigs.

OBJECTIVE AND DESIGN: This study evaluated the complement activation in guinea pigs that were given an intravenous injection of Sephadex and its correlation with markers of the development of inflammation. MATERIALS AND METHODS: Dunkin Hartley guinea pigs (250-300 g) were used. Whole blood was collected by heart puncture in a sodium citrate solution (0.315 g/ml) for complement measurements. Complement activation was measured using a colorimetric haemolytic assay. Bronchoalveolar lavages (BAL) were performed to monitor cell infiltration and inflammation was monitored by measurements of eosinophil peroxidase (EPO), histamine, beta-glucuronidase, albumin and total proteins in the BAL fluid. TREATMENT: Guinea pigs were pre-treated with aprotinin (40000 IKU/kg) 30 min before they were given an intravenous injection of Sephadex beads (24 mg/kg). Carboxymethyl (CM)-Sephadex (24 mg/kg) was administered alone. RESULTS: Sephadex beads activated the complement system in vitro (14.12+/-2.29 U/ml) and in vivo (9.95+/-0.08 U/ml) reaching a peak 6 h after the injection. This activation was accompanied by other characteristic features of inflammation such as leukocyte infiltration and activation. Both CM-Sephadex and aprotinin reduced the blood complement activation and eosinophil infiltration/activation observed. CONCLUSIONS: Our results strongly suggest that complement is involved in the cascade of events leading to the inflammatory state observed in guinea pig following the intravenous injection of Sephadex beads.

Selective inflammatory response induced by intratracheal and intravenous administration of poly-L-arginine in guinea pig lungs.

Major basic protein (MBP) is a cationic protein found in eosinophil granules that was postulated to participate in the pathogenesis of bronchial asthma. Recently, it has been demonstrated that MBP level in serum or bronchoalveolar lavage (BAL) fluid was correlated with bronchial hyperresponsiveness (BHR) in asthmatics. A number a studies have established that MBP actions could be mimicked by synthetic polycations as poly-L-arginine. In this study, we investigated the effects of intratracheal and intravenous administration of poly-L-arginine on lung inflammatory response development. The intratracheal injection of poly-L-arginine at the doses of 1, 10, 100 nmol/animal increased the number of eosinophils (up to 3.2 fold) and neutrophils (up to 12 fold) in BAL fluid. Eosinophil and neutrophil infiltration was reversed by 88% and 67% respectively following low molecular weight heparin treatment (500 microg/animal). The intravenous injection of increasing doses of poly-L-arginine (1, 10, 100, 500 nmol/animal) increased the number of eosinophils (up to 2.7 fold) but not neutrophil infiltration in guinea pig lungs. Eosinophil infiltration was reversed by 87% following low molecular weight heparin treatment (1.5 mg/animal). Intratracheal treatment with poly-L-arginine (100 nmol/animal) produced an important increase of beta-glucuronidase, histamine, eosinophil peroxidase (EPO) and albumin levels in BAL fluid, whereas the intravenous treatment (500 nmol/animal) did not. These results show that the route of administration of poly-L-arginine greatly influences its effect on inflammatory cell recruitment since both administration routes elicited eosinophil migration but only the intratracheal route stimulated the migration of neutrophils. Moreover, poly-L-arginine appeared to induce other inflammatory responses since it increased beta-glucuronidase, histamine, EPO and albumin levels in BAL fluid following intratracheal treatment. These results also showed that low molecular weight heparin significantly blocks the inflammatory responses elicited by poly-L-arginine.

Suitability of cell metabolic colorimetric assays for assessment of CD4+ T cell proliferation: comparison to 5-bromo-2-deoxyuridine (BrdU) ELISA.

The conventional tritiated thymidine (3H-TdR) incorporation assay is considered as the 'gold standard' for the assessment of cell growth. However, the 3H-TdR incorporation assay has several disadvantages which have prompted the development of nonradiolabelling proliferation assays such as 5-bromo-2-deoxyuridine (BrdU) ELISA, tetrazolium microplate assay and acid phosphatase assay. In studies, these three proliferation assays have shown equivalent sensitivity and reproducibility to the 3H-TdR incorporation assay. However the results may be affected by the cell type studied. In the present study, we have used these three proliferation assays for the assessment of rat lymph node CD4 + T lymphocyte growth in response to polyclonal antigen stimulation. The proliferation assays were compared on the basis of four criteria: sensitivity, reproducibility, stimulation index and insensitivity of the assay to the cell number. Our results indicated that the BrdU ELISA demonstrated the highest sensitivity, reproducibility and stimulation index but had a limited linear range for cellular growth. The tetrazolium microplate assay also had a relatively good sensitivity, reproducibility, stimulation index and a wider linear response range for cell growth in comparison to the BrdU ELISA. The acid phosphatase assay showed the lowest reproducibility and stimulation index. Because BrdU incorporation in DNA of proliferating cells has been reported to block cell division, we have investigated this possibility in sequential assays. Our results indicated that in our experimental conditions no evidence of an anti-mitogenic action of BrdU was observed. We also compared the performance of the MTS assay and BrdU ELISA in measuring substance P-induced CD4 + T cell proliferation. The results indicated that the MTS assay may reflect change in cell activation leading to an overestimate of cell growth. In conclusion, our results indicate that the BrdU ELISA is the most sensitive of the three proliferation assays used for the assessment of CD4 + T lymphocyte growth and is the preferred assay when small changes in cell growth are expected.

Involvement of alpha-4 integrins in allergic airway responses and mast cell degranulation in vivo.

Antibodies against integrins have been shown to inhibit allergic airway responses. The purpose of this study was to test the hypothesis that the beta1 integrin, very late antigen-4 (VLA-4), is involved in mast cell activation triggered by allergen exposure in sensitized animals. To do this we studied Brown Norway rats that were sensitized to ovalbumin (OA; 1 mg subcutaneously) using Bordetella pertussis as an adjuvant. Two weeks later rats were challenged with OA, pulmonary resistance (RL) was determined, and the concentrations of histamine and tryptase in bronchoalveolar lavage fluid and N-acetyl-leukotriene (LT)E4 in bile were measured. Pretreatment with a monoclonal antibody against VLA-4 (TA-2) attenuated the early response after OA challenge (342.9 +/- 24.4% baseline RL versus 153.3 +/- 19.4%; p < 0.01). There were significantly lower concentrations of histamine (67.11 +/- 11.90 microgram/ml versus 26.69 +/- 1.84; p < 0.01) and tryptase (0.143 +/- 0. 035 microgram/ml versus 0.053 +/- 0.022 microgram/ml; p < 0.01) in TA-2-treated animals. The increases in the concentrations of biliary N-acetyl-LTE4 after OA challenge were also significantly lower in TA-2-treated animals. These data suggest that a selective anti-VLA-4 monoclonal antibody prevents early responses through inhibition of mast cell activation.

Natural killer and lectin-dependent cytotoxic activities of Kurloff cells: target cell selectivity, conjugate formation, and Ca++ dependency.

Kurloff cells may represent a major component of NK cell activity in the guinea pig. We have pursued to characterize the mechanism of their action. Using murine target cells, we found Kurloff cell cytotoxicity to be selective for the NK-sensitive YAC-1 target cell, with minimal activity against the NK-resistant P815 target cell. In the presence of PHA, but not ConA, cytotoxicity was markedly augmented against both YAC-1 and P815. While effector-target conjugate formation was observed with YAC-1 cells but not P815 cells in control cultures, it was augmented with both target cell types in cultures with PHA. Pretreatment alone with PHA was ineffective, however. NK cell activity of Kurloff cells was dependent on extracellular Ca++ and entry of Ca++ into the effector cells, as demonstrated by abrogation of cytotoxicity when extracellular Ca++ was chelated with EDTA or EGTA, or following treatment with the Ca++ channel blockers verapamil and diltiazem. Furthermore, inhibition of PKC by H7 resulted in significant reduction of Kurloff cell-mediated NK activity, while pretreatment of effector cells with the PKC activator TPA enhanced NK activity. Kurloff cells could also be stimulated to produce serine esterases by contact with target cells or treatment with phorbol ester and ionophore. Finally, a majority of Kurloff cells, identified by the monoclonal antibody 14D1, reacted with the human NK cell marker CD56. Taken together, these data suggest that Kurloff cells have NK-like characteristics and activity, with target cell selectivity, and that their lytic mechanisms involve influx of extracellular Ca++, PKC activation and serine esterase production.

Kinetics of eosinophilia and eosinophil activation in the development of non-allergic bronchial hyperresponsiveness in guinea pigs injected with Sephadex beads.

Eosinophils are believed to play a crucial role in the pathogenesis of airway hyperresponsiveness (AHR). In the present study, the involvement of blood and pulmonary eosinophilia as well as the eosinophil activation in the onset of non-allergic AHR caused by the injection of G-50 Sephadex beads in guinea pigs was investigated. Reactivity of the isolated lower bronchus to histamine was measured ex vivo in a bioassay system. The increase of reactivity of the isolated lower bronchus of Sephadex-injected animals to histamine was observed as early as 3 h after the Sephadex injection and was maximal between 6-24 h. Sephadex-induced blood eosinophilia was characterized by two successive increases of blood eosinophil counts peaking at 3 and 12 h respectively. The recruitment of inflammatory cells into the lungs as measured in bronchoalveolar lavage fluid (BALF) have shown that the neutrophils were initially increased at 3 h whereas the number of eosinophils increased only 6 h after the bead injection; both cell populations were maximal 24 h later. Eosinophil peroxidase (EPO) activity was used as a marker for the apparent number of eosinophils in airways and the degree of activation of eosinophils recovered in BALF. Results have shown that EPO activity in the lower bronchus of Sephadex-injected animals increased at 6 h, decreased at 12 h and was maximal 24 h later. The EPO activity recovered in BALF was maximal between 6 to 24 h after the bead injection in guinea pigs. Correlation between the number of eosinophils and the EPO activity in BALF suggests that BALF eosinophils have been activated and have degranulated in airways. Correlation studies also indicated that both Sephadex induced blood eosinophilia and eosinophil activation were associated to the development of AHR. In contrast, the increase of EPO activity in the lower bronchus and BALF eosinophilia were not correlated to the development of AHR in our model. In conclusion, our results suggest that Sephadex induced non-allergic AHR in guinea pigs could be related, at least in part, to blood eosinophilia and eosinophil activation. Whether blood, airway and BALF eosinophilia as well as eosinophil activation are relevant factors to determine the potential role of eosinophils in the pathogenesis of AHR is discussed.

Guinea pig Kurloff (NK-like) cells mediate TNF-dependent cytotoxic activity: analogy with NC effector cells.

Kurloff cells are mononuclear cells possessing a large cytoplasmic inclusion body specific to the guinea pig. In this report, we present strong evidence that Kurloff cells can mediate NC activity against tumor cells in addition to their previously reported NK activity. Using an 18 h 51Cr-release assay we have shown that Kurloff cells were highly effective in killing the TNF-sensitive WEHI 164 target cell line. Lower but significant cytotoxic activity was also observed after only 4 h. However, our results suggest a different mechanism of lysis in the 4 h and 18 h assay. Lysis of WEHI 164 target cells by Kurloff cells in the 4 h assay could be strongly increased in the presence of TPA alone or in combination with ionomycin whereas ionomycin alone was uneffective. In contrast, stimulation of Kurloff cells for 18 h with ionomycin alone or in combination with TPA could induce the release of TNF-like factor(s) as observed by the TNF bioassay using L-929 TNF-sensitive target cells. Release of TNF-like factor(s) could also be induced by stimulation with WEHI 164 target cells. Supernatants of Kurloff cells stimulated for 18 h with TPA + ionomycin were also highly cytotoxic against WEHI 164 target cells, but not against the TNF-resistant P815 target cell line. Pretreatment of these supernatants with antimurine TNF alpha antibodies could almost completely inhibit their cytotoxic activity against WEHI 164 target cells. In contrast, supernatants of Kurloff cells stimulated for only 4 h did not show any TNF-like activity against the L-929 target cell line and were not cytotoxic against WEHI 164 target cells even after 18 h. Taken together, these results suggest that Kurloff cells can mediate NC activity against tumor cells in addition to their previously reported NK activity. By using multiple lytic pathways, these cells may play a crucial role in anti-tumor surveillance and defenses.