Tissue Culture Supernatants Research Articles

Simultaneous quantification of proinflammatory cytokines in human plasma using the LabMAP™ assay

There is an emerging trend in the pharmaceutical industry to evaluate a variety of surrogate biomarkers in Phase I/II clinical studies with the intention of determining potential activity of drugs early in clinical development. A number of cytokines expressed in pathological conditions are currently being considered as potential surrogates of disease and/or drug activity. The quantitative measurement of such analytes (biomarkers) in biological fluids has traditionally been performed by bioassays, enzyme-linked immunosorbent assay (ELISA), ribonuclease protection assay (RPA) and polymerase chain reaction (PCR). Typically, these methods have been limited to the measurement of a single analyte, require large sample volume and are time and cost involved. The LabMAP™ (Luminex™) system has been previously used to quantify cytokines in tissue culture supernatants and in animal serum. In the present study, the LabMAP™ technology was used for quantifying for the first time, pro-inflammatory cytokines such as, tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β), IL-6 and IL-8 levels in lipopolysaccharide (LPS)-stimulated human plasma samples. Both single-cytokine and two-cytokine (biplexed) panel formats were evaluated and the performance in the two formats was compared. A detailed validation procedure for these determinations is described along with a side-by-side comparison with ELISA results. Our results indicate that the LabMAP™ system can be used to measure cytokine levels in LPS-stimulated human plasma samples and that the levels obtained by this technique are comparable with ELISA results. It is therefore feasible to use this optimized technology to detect and quantify cytokines and other potential biomarkers in a complex milieu such as human plasma in support of clinical studies.

Loss of tuberin, the tuberous-sclerosis-complex-2 gene product is associated with angiogenesis.

Tuberous sclerosis complex (TSC) is an autosomal dominantly inherited disorder associated with an alteration of the TSC2 tumor suppressor gene which encodes for the protein product tuberin. The disease is characterized by the development of hamartomas, e.g. cutaneous angiofibromas which consist of vascular cells, interstitial cells, and normal components of the skin. The Eker rat model, an animal model of inherited cancer, has been shown to carry a mutation of TSC2. Immunohistochemical analyses of human angiofibromas were performed using antibodies directed against tuberin and angiogenic growth factors. Proliferation of human dermal microvascular endothelial cells (HDMEC) was determined after incubation with the supernatants of TSC2 (+/+) and TSC2 (-/-) rat embryonic fibroblasts (REF) that were derived from the Eker strain. Loss of the expression of tuberin was observed in the interstitial cells of 13 of 39 angiofibromas. The expression of tuberin was retained in the vascular cells. In all analyzed angiofibromas, the angiogenic factors bFGF, PD-ECGF, VEGF and angiogenin were detected in the interstitial cells and/or vascular cells. Expression of PDGF-B and TGF-beta1 was weak. Tissue culture supernatants from TSC2 (-/-) REF stimulated the growth of HDMEC significantly more than supernatants from TSC2 (+/+) REF. A functional loss of tuberin may stimulate vascular growth.

Morphine inhibits mucosal antibody responses and TGF-beta mRNA in gut-associated lymphoid tissue following oral cholera toxin in mice.

In this study, we investigated the effect of morphine on the mucosal immune system using fragment cultures of ileal segments, Peyer's patches (PPs), and mesenteric lymph nodes. Mice were implanted s.c. with a morphine slow release pellet. Control groups received a naltrexone slow release pellet, a placebo pellet, or both a morphine and a naltrexone pellet. After 48 h, mice were orally immunized with cholera toxin (CT) and were boosted orally 1 wk later. Animals were sacrificed 1 wk after the booster immunization, and PPs, mesenteric lymph nodes, and ileal segments were cultured in 24-well plates for 12 days. Morphine resulted in a highly significant inhibition of CT-specific IgA and IgG production in fragment culture supernatants of all three tissues compared with placebo. Naltrexone blocked the reduction in Ab levels induced by morphine, indicating that the effect is opioid receptor mediated. Morphine did not significantly alter total IgA levels in any of the tissue culture supernatants. Morphine also inhibited CT-specific IgA and IgG levels in serum. By flow cytometry, morphine did not alter the lymphoid cell composition in PPs compared with placebo. The effect of morphine on TGF-beta, IL-5, and IL-6 mRNA expression in PPs and ileal segments was determined following oral immunization with CT. Morphine significantly decreased TGF-beta mRNA compared with that in the placebo group, and naltrexone blocked this effect. These results indicate that morphine inhibits Ag-specific IgA responses in gut-associated lymphoid tissue at least partially through the inhibition of TGF-beta, a putative IgA switch factor, in the gastrointestinal tract.

In vitro activation of feline immunodeficiency virus in ramified microglial cells from asymptomatically infected cats.

Intravenous infection of cats with feline immunodeficiency virus was used as a model system to study activation of virus replication in brain-resident microglial cells in vitro. Virus release by ramified microglial cells isolated from subclinically infected animals was detectable in cell-free tissue culture supernatant only by reverse transcription and nested PCR of gag-specific RNA sequences and not by virion-associated reverse transcriptase activity. In contrast, cocultivation of in vivo-infected microglial cells with mitogen-activated peripheral blood mononuclear cells (PBMC) regularly allows detection of high virus yields in cell-free tissue culture fluid. Besides uptake and multiplication of microglia-derived virus in PBMC, release of virus from microglia is stimulated by cell contact with PBMC. The data suggest that T lymphocytes patrolling the central nervous system could reactivate the semilatent state of lentiviruses in microglial cells in the course of clinically silent central nervous system infection.

Functional role of melanoma inhibitory activity in regulating invasion and metastasis of malignant melanoma cells in vivo.

MIA (melanoma inhibitory activity) has been previously isolated from the tissue culture supernatant of melanoma cell lines as an autoregulatory activity, inhibiting thymidine incorporation. However, subsequent analyses of melanocytic tumours in vivo have correlated enhanced MIA expression with progression of melanocytic tumours, conflicting with the idea that MIA acts as a tumour suppressor. To investigate the role of MIA in vivo, we have therefore generated a panel of stably transfected B16 cell clones secreting different amounts of MIA. The capacity of these cell clones to form lung metastases in syngeneic C57Bl6 mice was strictly correlated to the level of MIA secretion, but the clones did not differ with respect to their proliferation in vitro. In summary, we suggest that MIA plays a causal role in promoting the metastasis of malignant melanomas, involving inhibition of tumour cell attachment to extracellular matrix molecules within their local milieu.

Virally Induced Lytic Cell Death Elicits the Release of Immunogenic GRP94/gp96

Necrotic cell death yields the release of cellular components that can function in the initiation of cellular immune responses. Given the established capacity of the endoplasmic reticulum chaperone GRP94 (gp96) to elicit CD8(+) T cell activation, we have investigated the cellular fate and antigenicity of GRP94 in differing scenarios of cell death. Virally induced cell death or mechanical cell death, elicited by freeze/thaw treatment of cell suspensions, yielded GRP94 release into the extracellular space; apoptotic cell death occurring in response to serum deprivation did not elicit GRP94 release. To assess the antigenicity of GRP94 released following virally induced cell death (lethal infection of cells with rVV ES-OVA(Met258-265), a recombinant, ovalbumin epitope-expressing vaccinia virus) or mechanical cell death (freeze/thaw of ovalbumin-expressing cells), tissue culture supernatant fractions were pulsed onto antigen-presenting cells, and antigen re-presentation was assayed as activation of an ovalbumin-specific T cell hybridoma. For both cell death scenarios, released GRP94 elicited a dose-dependent, ovalbumin-specific, hybridoma activation. In contrast, calreticulin derived from rVV ES-OVA(Met258-265)-infected cell extracts did not stimulate B3Z activity. These data identify GRP94 as an antigenic component released upon pathological, but not apoptotic, cell death and provide an assay system for the identification of cellular components of related activity.

CD14 is expressed and released as soluble CD14 by human intestinal epithelial cells in vitro: lipopolysaccharide activation of epithelial cells revisited.

Human endothelial as well as epithelial cells were shown to respond to lipopolysaccharides (LPSs). However, the expression and release of CD14 by these so-called CD14-negative cells have not been studied in detail. We investigated three human intestinal epithelial cell lines (ECLs), SW-480, HT-29, and Caco-2, for their expression of CD14 and CD11c/CD18 as well as their responsiveness to endotoxins. Fluorescence-activated cell sorter analysis revealed no expression of CD11c/CD18, but there was low expression of membrane-bound CD14 on HT-29, Caco-2, and SW-480 ECLs. Both Western blotting and reverse transcription-PCR confirmed the CD14 positivity of all three intestinal ECLs. No substantial modulation of CD14 expression was achieved after 6, 8, 18, 24, and 48 h of cultivation with 10-fold serial dilutions of LPS ranging from 0.01 ng/ml to 100 microg/ml. Interestingly, soluble CD14 was found in the tissue culture supernatants of all three ECLs. Finally, only HT-29 and SW-480, and not Caco-2, cells responded to LPS exposure (range, 0.01 ng/ml to 100 microg/ml) by interleukin 8 release. Thus, we show that HT-29, SW-480, and Caco-2 human intestinal ECLs express membrane-bound CD14. As Caco-2 cells did not respond to LPS, these cell lines might be an interesting model for studying the receptor complex for LPS. The fact that human intestinal epithelial cells are capable not only of expression but also of release of soluble CD14 may have important implications in vivo, e.g., in shaping the interaction between the mucosal immune system and bacteria in the gut and/or in the pathogenesis of endotoxin shock.

New high-performance liquid chromatographic method for the detection of picolinic acid in biological fluids

A HPLC method is described to quantify picolinic acid in milk, blood serum and tissue culture supernatant. The method requires very little sample preparation because acid precipitation allows total recovery of picolinic acid. High specificity and sensitivity were obtained using ion-pair chromatography on a C 18 reversed-phase column with tetrabutylammonium hydrogen sulfate as ion pairing reagent. We describe the conditions for the automated testing of multiple samples and for the detection of l-tryptophan and l-kynurenine together with picolinic acid. This system will be utilized to elucidate the relationship between picolinic acid production and human disease. Furthermore, we provide the first evidence of picolinic acid in human blood serum.

Direct isolation of monoclonal antibodies from tissue culture supernatant using the cation-exchange cellulose Express-Ion S

The chromatography of the murine hybridoma cell C595/102 culture supernatant expressing the therapeutic monoclonal antibody C595, on the cation-exchange cellulose Whatman Express-Ion Exchanger S has been investigated. Initial method scouting studies using purified C595 in 1-ml mini columns demonstrated that binding capacity and binding efficiency were dependent not only on decreasing pH but also on the buffer salts used to prepare the mobile phase. Under optimised conditions of 0.1 M sodium acetate buffer, pH 5.0, we were able to separate purified C595 from BSA, the major contaminant in tissue culture fluid. Under these conditions immunoreactive C595 could be isolated directly from tissue culture supernatant. A scale-down study was carried out using a 25-ml column operated at a flow-rate of 150 cm/h which also yielded purified immunoreactive antibody. This procedure should now be suitable for scale-up.

Rotavirus survival and stability in foods as determined by an optimised plaque assay procedure

Tissue culture adapted rotavirus strains were propagated in MA104 and CaCo2 cells using standard cell culture procedures. The progress of infection was monitored by examining for a cytopathic effect, and for the presence of viral RNA in the tissue culture supernatant as determined by a guanidinium-based method. Subsequently, an effective plaque assay for rotavirus was developed using MA104 cells by optimising the adsorption time (2 h) and the levels of fetal calf serum (2.5%) in the overlay medium. Tragacanth gum was used in the overlay medium to immobilise the virus, and plaques were subsequently stained with 1% crystal violet. Using this optimised plaque assay, the survival of rotavirus following exposure to heat and UV irradiation was evaluated by enumerating the clear plaques. It was shown that 60°C for 10 min was sufficient to reduce the viral titer by at least 7 logs, and 50 mJ of UV irradiation was sufficient to reduce the initial viral titer by >2.5 logs. This optimised plaque assay was also used to determine the survival and stability of rotavirus from a range of experimentally contaminated foods including fruit juice, formula milk and lettuce.

Non-urease producing Helicobacter pylori in chronic gastritis.

Helicobacter pylori infection is the commonest cause of gastritis. Different patterns of immune response to H. pylori infection and characteristics of bacteria are considered to contribute to clinical outcomes. To determine characteristics of the host H. pylori relationship in subjects with non-ulcer dyspepsia and a histological diagnosis of gastritis. Thirty-five subjects with chronic gastritis undergoing endoscopy (mean age 53 years, range 24-82, 14 male and 21 female) were studied, none of whom was on nonsteroidal anti-inflammatory drugs or antibiotics. H. pylori infection was determined by rapid urease test (CLOtest), culture, antibody and RT-PCR for Ure C, Cag A and 26 kDa gene and histology. Cytokine production of mucosal IL-6 and IL-8 were measured by ELISA. Fifteen subjects were positive by CLOtest and/or bacterial culture. In these subjects histology showed numerous helical forms of H. pylori (Group I). Nine subjects were negative by CLOtest, bacterial culture, and mRNA for urease C fragment, but positive by PCR for the 26 kDa protein encoding gene. Histology in these subjects showed the presence of either coccoid forms (four), or scant helical forms (two), or mixed coccoid/helical forms (three) (Group II). Eleven subjects were negative by all methods of detection (Group III). IgG and IgA antibody levels in serum (p<0.05) and gastric tissue culture supernatant (p<0.001) were significantly higher in Group I than those in Group II or III. There were significant differences in the IgG serum and IgA supernatant antibody levels (p<0.01 and p<0.05) when Group II was compared to Group III. Supernatant IL-6 levels were significantly higher in Group I (p<0.01) than those from Groups II and III. IL-8 levels were higher in Group I (p<0.01) and Group II (p<0.05) when compared to Group III. 'H. pylori-negative' gastritis can be associated with a non-urease producing form of H. pylori, with a reduction in both local and systemic antibody levels and mucosal pro-inflammatory cytokines.

Single-shot plasmid DNA intrasplenic immunization for the production of monoclonal antibodies: Persistent expression of DNA

Monoclonal antibodies (Mc. Abs.) were generated against a 18-kDa protein from Brucella abortus 48 h and 25 days after a single intrasplenic injection of a DNA plasmid containing the expression vector for the protein. Hybridomas were also obtained from spleens injected 3, 5, and 10 days before fusion. Somatic cell fusion of spleen cells from mice, injected with the plasmid DNA, in saline, with the NS-0 myeloma cell line resulted in Mc. Abs of the IgG and IgM Isotypes. IgG antibodies were of the IgG2b and IgG1 subtype. Hybridoma tissue culture supernatants were strongly positive by ELISA at dilutions of up to 1/1200 and produced intense specific bands in immunoblotting. All these antibodies recognized the native recombinant protein (the screening antigen) and some of them also recognized the heat-denatured recombinant 18-kDa protein. When compared to standard procedures of immunization, as well as to intramuscular or gene gun DNA immunizations, this technique results in very early, time saving, strong Mc Abs. It is common knowledge that in order to generate specific hybridomas; spleen cells from immunized animals have to be fused no later than 5 days after the last boost. The fact that through single-shot intrasplenic immunization (SSI) specific hybridomas are generated 25 days after one single injection indicates that the gene coding the p18 protein is being expressed in the spleen for at least 20 days. We propose that plasmid DNA intrasplenic immunization can be a helpful tool for the production of specific hybridomas. This route of immunization could also be helpful in the further understanding of early events of the immune response to genetic immunization by naked DNA injection.

Mucosal Production of Antigastric Autoantibodies in Helicobacter pylori Gastritis

Apart form bacterial virulence factors of Helicobacter pylori, certain host factors influence the pathogenesis of H. pylori gastritis. In particular, antigastric autoantibodies that are detectable in the sera of a substantial proportion of H. pylori were shown to correlate with the development of gastric atrophy. The aim of this study was to analyze the possible antigastric autoimmune response in H. pylori gastritis at the site where the action is, i.e. , in the gastric mucosa. Gastric biopsy specimens from antrum and corpus mucosa of 24 H. pylori-infected and of 33 noninfected patients were cultured for 3 days, and tissue culture supernatants were analyzed for the amount of locally produced IgA and IgG. Antigastric autoantibodies were screened in the sera and in the supernatants by means of immunohistochemistry. The infected patients had significantly higher concentrations of locally produced IgA, whereas the IgG concentrations were virtually the same in infected and noninfected patients. IgG or IgA antigastric autoantibodies, or both, were detectable only in the sera (38%) and supernatants (17%) of infected patients. Interestingly, the patient with the strongest local autoimmune response showed body-predominant H. pylori gastritis, with destruction of gastric glands and atrophy of the body mucosa. These results demonstrate that antigastric autoimmune reactions are detectable at the site of the disease and might be relevant for the pathogenesis of gastric mucosa atrophy in H. pylori gastritis.

Cloning and Expression of Secretagogin, a Novel Neuroendocrine- and Pancreatic Islet of Langerhans-specific Ca2+-binding Protein

We have cloned a novel pancreatic beta cell and neuroendocrine cell-specific calcium-binding protein termed secretagogin. The cDNA obtained by immunoscreening a human pancreatic cDNA library using the recently described murine monoclonal antibody D24 contains an open reading frame of 828 base pairs. This codes for a cytoplasmic protein with six putative EF finger hand calcium-binding motifs. The gene could be localized to chromosome 6 by alignment with GenBank genomic sequence data. Northern blot analysis demonstrated abundant expression of this protein in the pancreas and to a lesser extent in the thyroid, adrenal medulla, and cortex. In addition it was expressed in scant quantity in the gastrointestinal tract (stomach, small intestine, and colon). Thyroid tissue expression of secretagogin was restricted to C-cells. Using a sandwich capture enzyme-linked immunosorbent assay with a detection limit of 6.5 pg/ml, considerable amounts of constitutively secreted protein could be measured in tissue culture supernatants of stably transfected RIN-5F and dog insulinoma (INS-H1) cell clones; however, in stably transfected Jurkat cells, the protein was only secreted upon CD3 stimulation. Functional analysis of transfected cell lines expressing secretagogin revealed an influence on calcium flux and cell proliferation. In RIN-5F cells, the antiproliferative effect is possibly due to secretagogin-triggered down-regulation of substance P transcription.

Steroid hormone activity of flavonoids and related compounds.

Soy isoflavones have been studied extensively for estrogenic and antiestrogenic properties. Other flavonoids, found in fruits, vegetables, tea and wine, have been much less tested for steroid hormone activity. We therefore assessed the estrogenic, androgenic and progestational activities of 72 flavonoids and structurally-related compounds. These compounds were tested on BT-474 human breast cancer cells at concentrations of 10(8)-10(-5) M, with estradiol (estrogen), norgestrel (progestin) and dihydrotestosterone (androgen) used as positive controls, and ethanol (solvent) as a negative control. pS2, an estrogen-regulated protein, and prostate-specific antigen (PSA), regulated by androgens and progestins, were quantified in tissue culture supernatants using ELISA-type immunofluorometric assays developed in-house. Of the 72 compounds tested, 18 showed estrogenic activity at 10(-5) M. Of this subset, seven also showed progestational activity at this concentration. The soy isoflavones, biochanin A and genistein, showed the most potent estrogenic activity, with a dose-response effect up to 10(-7) M. Of all other flavonoids, luteolin and naringenin displayed the strongest estrogenicity, while apigenin had a relatively strong progestational activity. Based on our data, we have formulated a set of structure/function relationships between the tested compounds. Flavonoids, therefore, exhibit significant steroid hormone activity, and may have an effect in the modification of cancer risk by diet, or in cancer therapeutics and prevention.

Angiogenesis Induced by the Implantation of Self-Bone Marrow Cells: A New Material for Therapeutic Angiogenesis

Bone marrow, contains various primitive cells that are thought to secrete several angiogenic growth factors and may also differentiate into endothelial cells. The present study was conducted to investigate the possibility that bone marrow cells could be a novel material to induce angiogenesis. The expression of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) in rat bone marrow cells was examined by immunohistochemistry. The production of VEGF was compared in tissue culture supernatant under the conditions of normoxia and hypoxia. The process of angiogenesis that occurred following the implantation of bone marrow cells was determined using a rat cornea model. VEGF- and bFGF-positive cells were found in rat bone marrow. The production of VEGF from bone marrow cells was significantly more enhanced by hypoxic conditions than by normoxic conditions. The rat cornea model showed that bone marrow cell implantation created new vessels. The implantation of self-bone marrow cells is a novel and simple method of inducing angiogenesis.

Mucosal humoral immune response to CagA shows a high prevalence in patients with gastric MALT-type lymphoma.

In the pathogenesis of gastric mucosa-associated lymphoid tissue (MALT)-type lymphoma, CagA-positive Helicobacter pylori strains have been suspected of making a significant contribution. To investigate this hypothesis in more detail, the mucosal humoral immune response of 15 patients with gastric MALT-type lymphoma was examined in the tumor and in the tumor-free gastritis of the same patient. Mononuclear cells from different sites (antrum, corpus, lymphoma) were cultured. Culture supernatant and serum of the same patient were used for immunodetection of CagA. All patients displayed an immune response to CagA in the tissue-culture supernatants. Although the humoral immune response in the tumor was restricted to a very few H. pylori antigens, antibodies directed against CagA protein were found in most patients. The immune response to CagA in nearly all lymphoma patients--not only in the serum, but also in the mucosa, including the tumor site--support the hypothesis that CagA is involved in the pathogenesis of gastric MALT-type lymphoma.

Production and functional characterization of a soluble recombinant form of mouse CD59.

This report describes the engineering, expression, purification and functional characterization of a soluble recombinant form of murine CD59 (srMoCD59). We report the expression in Chinese hamster ovary (CHO) cells of a modified mouse CD59 cDNA that had been truncated at D-74, resulting in the loss of the glycosylphosphatidyl inositol (GPI) anchor, and containing six additional C-terminal histidines. The expressed srMoCD59 was purified from tissue culture supernatant by means of its poly-histidine tag using immobilized metal affinity chromatography. In comparison with CD59 on mouse erythrocytes, the srMoCD59 had a reduced molecular weight (18-20 000 as compared with 20-28 000 for GPI-anchored srMoCD59). The terminal complement inhibitory capacity of this soluble recombinant protein was assessed using two methods: a cobra venom factor (CVF)-triggered 'reactive-lysis' system and a C5b-7 site assay. In both assays, srMoCD59 inhibited lysis by the sera from all three species tested in the rank order mouse > rat >> human. The amount of srMoCD59 required to produce 50% inhibition of lysis in the C5b-7 site assay, using purified terminal components to develop lysis, was 10-fold less than that required in the same assay when EDTA serum was used as a source of C8 and C9, or in the CVF reactive lysis system. These data indicate that the presence of serum markedly interfered with the activity of srMoCD59 and have important implications for the use of recombinant soluble CD59 analogues as therapeutic agents in complement-mediated diseases.

Differential steroid hormone regulation of human glandular kallikrein (hK2) and prostate-specific antigen (PSA) in breast cancer cell lines.

We have investigated the steroid hormone regulation of human glandular kallikrein (hK2) and prostate-specific antigen (PSA) in the breast cancer cell lines BT-474, T-47D, MFM-223, MCF-7, ZR-75-1, MDA-MB-435, and BT-20. Using highly sensitive time-resolved fluorometric immunoassays, we were able to detect significant amounts of both kallikreins in tissue culture supernatants of BT-474, T-47D, and MFM-223 cells after hormonal stimulation. However, BT-474 cells produce much more hK2 than PSA, whereas the situation is reversed in T-47D cells. Furthermore, BT-474 cells produce, on absolute terms, about 500-1,000-fold more hK2 than T-47D cells. From all steroids tested, mibolerone, a synthetic non-metabolizable androgen, was the most potent stimulator for both kallikreins followed by the synthetic progestin norgestrel. Estradiol was able to induce production of small but significant amounts of hK2 and PSA in the BT-474 cell line, supporting the notion that there is a cross-talk between the estrogen and androgen hormone-receptor signaling pathways. MFM-223 is an androgen responsive cell line, devoid of other steroid hormone receptors, which is also capable of producing hK2 and PSA but at much lower amounts. MCF-7 and ZR-75-1 cell lines failed to produce any protein, even though they have similar steroid receptor content as the BT-474 and T-47D cell lines. This was also the case for MDA-MB-435, a cell line rich in androgen receptors. Our data suggest that the expression of the hK2 gene in breast cancer cell lines is mainly under the control of androgens and progestins, similarly to PSA. These cell lines may represent good models for studying the differential expression of these two genes and for identifying cellular factors (e.g. co-activators/co-repressors), which may modify the potency of expression after hormonal stimulation.

MULTIPLE MODES OF RESPONSES TO AIR POLLUTION PARTICULATE MATERIALS IN A549 ALVEOLAR TYPE II CELLS

Exposure to components of air pollution may cause adverse effects on lung cellular and organ functions through several mechanisms. Cell death, altered gene expression including production of cytokines, and modifications of normal cellular processes are possible outcomes that may be independent or coupled. To assess the effects of materials representative of a variety of particulate components of air pollution on lung epithelium, a human cell line of type II origin (A549 cells) was exposed to these materials in vitro. Materials tested included carbon black (CB), diesel soot from two sources (DS), residual oil fly ash (ROFA), Ottowa Ambient Air particulate (OAA), silicon dioxide (SiO2), and nickel subsulfide (Ni3S2). Endpoints included loss of adherence measured by crystal violet staining (CV), lactate dehydrogenase release (LDH), release of interleukin-8 (IL-8) measured by ELISA, and alkaline phosphatase activity in the cells (APc) and released into the supernatant (APs). Nuclear morphology was also examined. SiO2 and Ni3S2 both caused dose-dependent acute toxicity as assessed by LDH and CV,and caused alterations in nuclear morphology consistent with apoptosis. However, much more IL-8 was released into the tissue culture supernatant by SiO2 at the same levels of cytotoxicity than by Ni3S2. Neither of these acutely toxic materials increased APc or APs, but the less cytotoxic materials caused very significant release of AP in the order OAA>DS>ROFA>> SiO2 = Ni3S2. OAA and, to a lesser extent, DS caused increases in mitotic fraction and increased CV staining, consistent with stimulation of proliferation. These results suggest multiple modes of responses to toxic materials and imply that a toxicological screening process should address these and possibly other endpoints.