Joseph K. H. Ma

The Transport Barrier of Epithelia: A Comparative Study on Membrane Permeability and Charge Selectivity in the Rabbit

The transport barrier of the epithelia presents one of the major problems limiting the effective use of these tissues as alternate delivery routes for macromolecules such as peptides and proteins. In the present study, two membrane transport properties, namely, the permeability and permselectivity of the shunt pathway, were investigated and compared in various tissues including the nasal, tracheal, bronchial, buccal, rectal, vaginal, corneal, epidermal, duodenal, jejunal, ileal, and colonic epithelia. Membrane permeability was evaluated using a combined method based on electrical conductance and flux measurements of a hydrophilic fluorescent probe, 6-carboxy fluorescein (CF). Membrane permselectivity or the charge discriminating ability of the membrane was evaluated by KCl diffusion potential measurements. The results indicate that all epithelia under investigation possess a relatively high degree of permeation barrier and are highly selective for the absorption of positively charged solutes. Shunt path permeability was found to vary greatly among tissues from different epithelia, whereas membrane charge selectivity was relatively constant in these tissues. A good correlation was observed between membrane electrical conductance and steady-state flux of CF, indicating a paracellular transport of the compound. The rank order of the intrinsic membrane permeability was as follows: intestinal≈ nasal ≥ bronchial ≥ tracheal > vaginal ≥ rectal > corneal > buccal > skin. Membrane permselectivity, expressed as the ratio of transport number (positive over negative), ranges from 1.78 for the buccal to 1.33 for the rectal epithelium. These results suggest that, for effective delivery purposes, permeation enhancing methods, by either increasing tissue permeability or modifying drug-membrane charge selectivity, are generally required. The permeation data also suggest that the respiratory epithelia represent good alternate routes for drug delivery, particularly for those that are orally ineffective, i.e., due to extensive gastrointestinal tract degradation or first-pass metabolism.

THE DUAL EFFECT OF THE PARTICULATE AND ORGANIC COMPONENTS OF DIESEL EXHAUST PARTICLES ON THE ALTERATION OF PULMONARY IMMUNE/INFLAMMATORY RESPONSES AND METABOLIC ENZYMES

ABSTRACT Exposure to diesel exhaust particles (DEP) is an environmental and occupational health concern. This review examines the cellular actions of the organic and the particulate components of DEP in the development of various lung diseases. Both the organic and the particulate components cause oxidant lung injury. The particulate component is known to induce alveolar epithelial damage, alter thiol levels in alveolar macrophages (AM) and lymphocytes, and activate AM in the production of reactive oxygen species (ROS) and pro-inflammatory cytokines. The organic component, on the other hand, is shown to generate intracellular ROS, leading to a variety of cellular responses including apoptosis. There are a number of differences between the biological actions exerted by these two components. The organic component is responsible for DEP induction of cytochrome P450 family 1 enzymes that are critical to the polycyclic aromatic hydrocarbons (PAH) and nitro-PAH metabolism in the lung as well as in the liver. The particulate component, on the other hand, causes a sustained down-regulation of CYP2B1 in the rat lung. The significance of this effect on pulmonary metabolism of xenobiotics and endobiotics remains to be seen, but may prove to be an important factor governing the interplay of the pulmonary metabolic and inflammatory systems. Long-term exposures to various particles including DEP, carbon black (CB), TiO2, and washed DEP devoid of the organic content, have been shown to produce similar tumorigenic responses in rodents. There is a lack of correlation between tumor development and DEP chemical-derived DNA adduct formation. But the organic component has been shown to generate ROS that produce 8-hydroxydeoxyguanosine (8-OHdG) in cell culture. The organic, but not the particulate, component of DEP suppresses the production of pro-inflammatory cytokines by AM and the development of Th1 cell-mediated immunity. The mechanism for this effect is not yet clear, but may involve the induction of heme oxygenase-1 (HO-1), a cellular genetic response to oxidative stress. Both the organic and the particulate components of DEP enhance respiratory allergic sensitization. Part of the DEP effects may be due to a depletion of glutathione in lymphocytes. The organic component, which is shown to induce IL-4 and IL-10 productions, may skew the immunity toward Th2 response, whereas the particulate component may stimulate both the Th1 and Th2 responses. In conclusion, the literature shows that the particulate and organic components of DEP exhibit different biological actions but both involve the induction of cellular oxidative stress. Together, these effects inhibit cell-mediated immunity toward infectious agents, exacerbate respiratory allergy, cause DNA damage, and under long-term exposure, induce the development of lung tumors.

Cerium oxide nanoparticle-induced pulmonary inflammation and alveolar macrophage functional change in rats

Abstract The use of cerium compounds as diesel fuel catalyst results in the emission of cerium oxide nanoparticles (CeO2) in the exhaust. This study characterized the potential effects of CeO2 exposure on lung toxicity. Male Sprague Dawley rats were exposed to CeO2 by a single intratracheal instillation at 0.15, 0.5, 1, 3.5 or 7 mg/kg body weight. At 1 day after exposure, CeO2 significantly reduced NO production, but increased IL-12 production, by alveolar macrophages (AM) in response to ex vivo lipopolysacchride (LPS) challenge, and caused AM apoptosis, through activation of caspases 9 and 3. CeO2 exposure markedly increased suppressor of cytokine signaling-1 at 1-day and elevated arginase-1 at 28-day post exposure in lung cells, while osteopontin was significantly elevated in lung tissue at both time points. CeO2 induced inflammation, cytotoxicity, air/blood barrier damage, and phospholipidosis with enlarged AM. Thus, CeO2 induced lung inflammation and injury in lungs which may lead to fibrosis.

Effects of Diesel Exhaust Particles on the Release of Interleukin-1 and Tumor Necrosis Factor-Alpha from Rat Alveolar Macrophages

The effects of diesel exhaust particles (DEP) and their components (washed dust and methanol extracts) on the release of proinflammatory cytokines, interleukin-I (IL-1), and tumor necrosis factor-alpha (TNF-alpha) by alveolar macrophages (AM) were investigated. Rat AM were incubated with 0, 5, 10, 20, 50, or 100 micrograms/10(6) AM/mL of DEP, methanol-washed DEP, or equivalent concentrations of DEP methanol extracts at 37 degrees C for 24 h. AM-conditioned supernatants were collected and assayed for the activities of IL-1 and TNF-alpha. At high concentrations both DEP and DEP methanol extracts were shown to increase IL-I-like activity secreted by AM, whereas methanol-washed DEP had no effect. Neither DEP, methanol-washed DEP, nor DEP methanol extracts was found to stimulate the secretion of TNF-alpha. The effects of DEP on the release of IL-I and TNF-alpha by lipopolysaccharide (LPS)- or interferon-gamma (IFN-gamma)-primed AM were also studied. AM were preincubated with various concentrations of DEP for 2 h, then challenged with either 0.1 microgram/mL of LPS or 5 units/mL of IFN-gamma. DEP inhibited LPS-stimulated production of H-I and TNF-alpha. These inhibitory effects were attributed to the organic extracts of DEP. In contrast, stimulation of AM production of TNF-alpha by IFN-gamma was not affected by DEP exposure. In summary, evidence that DEP enhanced the production of IL-1 by AM in vitro suggests that this proinflammatory cytokine may play a role in the pulmonary response to DEP inhalation. The suppressive response of DEP-pretreated AM to LPS stimulation may be a contributing factor to the impairment of pulmonary defense system after prolonged DEP exposure.

EFFECTS OF DIESEL EXHAUST PARTICLES (DEP), CARBON BLACK, AND SILICA ON MACROPHAGE RESPONSES TO LIPOPOLYSACCHARIDE: EVIDENCE OF DEP SUPPRESSION OF MACROPHAGE ACTIVITY

The effects of diesel exhaust particle (DEP) exposure on alveolar macrophage (AM) response to ex vivo and in vivo lipopolysaccharide (LPS) challenge were determined by monitoring LPS-stimulated production of interleukin-1 (IL-1) and tumor necrosis factor-alpha (TNF-alpha). The roles of the insoluble particulate and the organic compounds of DEP in altering pulmonary responses were evaluated by comparing the DEP-induced pulmonary responses to those of carbon black (CB), a carbonaceous particle with few adsorbed organic compounds, or to silica, a known pneumotoxic dust. Male Sprague-Dawley rats were exposed to a single intratracheal dose (5 or 35 mg/kg body weight) of DEP, CB, or silica, or to saline vehicle. Rats were sacrificed 1, 3, or 7 d postexposure. To study the responsiveness to the bacterial product LPS, AM isolated from particle-exposed rats were challenged ex vivo with LPS (0.1 microg/10(6) AM) and LPS-stimulated cytokine release was monitored. In addition, rats were exposed intratracheally to a single dose of DEP (5 mg/kg) and 3 d later exposed in vivo to 1 mg/kg LPS for 3 h prior to measurement of cytokine production by AM. DEP exposure resulted in neutrophil infiltration and elevated levels of albumin and lactate dehydrogenase (LDH) activity in the bronchoalveolar lavage fluid; these responses were not substantially different from those elicited by CB or silica exposure. AM from DEP-exposed rats showed increased spontaneous production of IL-1, but not TNF-alpha, while the opposite was true for CB or silica. Upon ex vivo challenge with LPS, AM from DEP-exposed rats showed a significant decrease in the secretion of TNF-alpha and, to a lesser extent, IL-1, compared to the sum of the DEP and LPS effects. In contrast, AM from CB- or silica-exposed rats did not show this decreased responsiveness to subsequent LPS challenge. This inhibitory action of DEP on LPS-stimulated AM production of IL-1 and TNF-alpha was further confirmed by the results obtained from rats exposed to both DEP and LPS in vivo. In summary, these results indicate that while DEP, CB, and silica all induce pulmonary inflammatory responses due to particle stimulation, only DEP suppress AM cytokine release in response to LPS stimulation. The contrasting cellular response with respect to DEP and CB exposures may be due to the presence of adsorbed organic compounds on DEP, which may contribute to the increased susceptibility of hosts to pulmonary infections after DEP exposure.

Induction of pulmonary fibrosis by cerium oxide nanoparticles

Cerium compounds have been used as a diesel engine catalyst to lower the mass of diesel exhaust particles, but are emitted as cerium oxide (CeO(2)) nanoparticles in the diesel exhaust. In a previous study, we have demonstrated a wide range of CeO(2)-induced lung responses including sustained pulmonary inflammation and cellular signaling that could lead to pulmonary fibrosis. In this study, we investigated the fibrogenic responses induced by CeO(2) in a rat model at various time points up to 84 days post-exposure. Male Sprague Dawley rats were exposed to CeO(2) by a single intratracheal instillation. Alveolar macrophages (AM) were isolated by bronchial alveolar lavage (BAL). AM-mediated cellular responses, osteopontin (OPN) and transform growth factor (TGF)-β1 in the fibrotic process were investigated. The results showed that CeO(2) exposure significantly increased fibrotic cytokine TGF-β1 and OPN production by AM above controls. The collagen degradation enzymes, matrix metalloproteinase (MMP)-2 and -9 and the tissue inhibitor of MMP were markedly increased in the BAL fluid at 1 day- and subsequently declined at 28 days after exposure, but remained much higher than the controls. CeO(2) induced elevated phospholipids in BAL fluid and increased hydroxyproline content in lung tissue in a dose- and time-dependent manner. Immunohistochemical analysis showed MMP-2, MMP-9 and MMP-10 expressions in fibrotic regions. Morphological analysis noted increased collagen fibers in the lungs exposed to a single dose of 3.5mg/kg CeO(2) and euthanized at 28 days post-exposure. Collectively, our studies show that CeO(2) induced fibrotic lung injury in rats, suggesting it may cause potential health effects.

Effect of diesel exhaust particulate (DEP) on immune responses: contributions of particulate versus organic soluble components

The effect of diesel exhaust particulate (DEP) exposure on innate, cellular and humoral pulmonary immunity was studied using high-dose, acute-exposure rat, mouse, and cell culture models. DEP consists of a complex mixture of petrochemical-derived organics adsorbed onto elemental carbon particles. DEP is a major component of particulate urban air pollution and a health concern in both urban and occupational environments. The alveolar macrophage is considered a key cellular component in pulmonary innate immunity. DEP and DEP organic extracts have been found to suppress alveolar macrophage function as demonstrated by reduced production of cytokines (interleukin-1 [IL-1], tumor necrosis factor-α[TNF-α]) and reactive oxygen species (ROS) in response to a variety of agents, including lipopolysaccharide (LPS), interferon-γ(IFN-γ), and bacteria. Fractionation of DEP organic extract suggests that this activity was predominately in polyaromatic-containing and more polar (resin) fractions. Organic-stripped DEP did not alter these innate pulmonary immune responses. DEP also depressed pulmonary clearance of Listeria monocytogenes and Bacillus Calmette-Guerin (BCG). The contribution of the organic component of DEP is less well defined with respect to acquired and humoral immunity. Indeed, both DEP and carbon black enhanced humoral immune responses (specific immunoglobulin [Ig] E and IgG) in an ovalbumin-sensitized rat model. It is concluded that both the particulate and adsorbed organics may contribute to DEP-mediated immune alterations.

Regulation of tight junction permeability by calcium mediators and cell cytoskeleton in rabbit tracheal epithelium

The present study investigates the mechanisms controlling tight junction permeability of the tracheal epithelium, with an emphasis on the regulatory role of intra- and extracellular calcium as well as the cell cytoskeleton. The tracheas were isolated from rabbits and their junctional permeability barrier was investigated in vitro by means of transepithelial electrical resistance measurements and flux measurements of the radiolabeled paracellular tracer, 14C-mannitol. The effects of intra- and extracellular calcium were studied using the calcium ionophore A 23187 and EGTA, and that of the cytoskeleton was investigated using cytochalasin B. Intracellular calcium of the tracheal epithelium was monitored microfluorometrically using the specific calcium indicator, Fura-2 AM (acetoxymethyl ester). The results indicate that the tight junction permeability of the trachea was significantly increased upon treatment with all three of the test compounds, as evidenced by a substantial decrease in transepithelial electrical resistance and an increase in transepithelial flux of 14C-mannitol. The effects of EGTA and cytochalasin B on the tight junction permeability are fully reversible upon removal of the compounds from the bathing media. On the other hand, tissues treated with the calcium ionophore demonstrate a partial or no recovery in membrane permeability, depending on the intracellular calcium levels. Moderate and transient increases in intracellular calcium caused a partial reversibility of the membrane resistance, while high and sustained intracellular calcium levels induce a complete irreversibility of the membrane resistance. These results suggest that high extracellular calcium levels and low intracellular calcium levels are required for the normal maintenance of the junctional permeability in the tracheal epithelium. Studies using cytochalasin B indicate that there is also a close relationship between the tight junctions and the organization of actin microfilaments. Alterations of these structures as well as cellular calcium levels can result in a substantial change in transepithelial permeability. Therefore compounds that affect tight junction permeability may exert their action through the calcium and cytoskeleton mechanisms.

Reactive oxygen species- and nitric oxide-mediated lung inflammation and mitochondrial dysfunction in wild-type and iNOS-deficient mice exposed to diesel exhaust particles.

Pulmonary responses to diesel exhaust particles (DEP) exposure are mediated through enhanced production of reactive oxygen species (ROS) and nitric oxide (NO) by alveolar macrophages (AM). The current study examined the differential roles of ROS and NO in DEP-induced lung injury using C57B/6J wild-type (WT) and inducible NO synthase knockout (iNOS KO) mice. Mice exposed by pharyngeal aspiration to DEP or carbon black particles (CB) (35 mg/kg) showed an inflammatory profile that included neutrophil infiltration, increased lactate dehydrogenase (LDH) activity, and elevated albumin content in bronchoalveolar lavage fluid (BALF) at 1, 3, and 7 d postexposure. The organic extract of DEP (DEPE) did not induce an inflammatory response. Comparing WT to iNOS KO mice, the results show that NO enhanced DEP-induced neutrophils infiltration and plasma albumin content in BALF and upregulated the production of the pro-inflammatory cytokine interleukin 12 (IL-12) by AM. DEP-exposed AM from iNOS KO mice displayed diminished production of IL-12 and, in response to ex vivo lipopolysaccharide (LPS) challenge, decreased production of IL-12 but increased production of IL-10 when compared to cells from WT mice. DEP, CB, but not DEPE, induced DNA damage and mitochondria dysfunction in AM, however, that is independent of cellular production of NO. These results demonstrate that DEP-induced immune/inflammatory responses in mice are regulated by both ROS- and NO-mediated pathways. NO did not affect ROS-mediated mitochondrial dysfunction and DNA damage but upregulated IL-12 and provided a counterbalance to the ROS-mediated adaptive stress response that downregulates IL-12 and upregulates IL-10.

Targeted gene delivery to alveolar macrophages via Fc receptor-mediated endocytosis

Alveolar macrophage (AM) plays important roles in lung homeostasis and pathogenesis of diseases. The study of macrophage gene function and regulation as well as its potential therapeutic intervention will require the development of vectors capable of safe and efficient transfer of DNA to the AM. In the present study, we report a new transfection system that utilizes Fc receptor-mediated endocytosis as a means to target DNA to the AM. This system employs molecular conjugates consisting of a cognate moiety, in this case IgG which recognizes the AM Fc receptor, covalently-linked to a DNA-binding moiety, such as a cationic polyamine. A Complex was formed between immunoglobulin G-polylysine conjugate (IgG-pL) and plasmid DNA carrying the LacZ reporter gene (pSVβ). The conjugate-DNA complex was added directly to the AMs in culture and incubated for 24 h, after which LacZ gene expression was analyzed for β-galactosidase activity by microfluorometry using a fluorogenic β-galactosidase substrate, 5-dodecanoylaminofluorescein di-β-D-galactopyranoside (C12FDG). The AMs treated with the IgG-pL/DNA complex exhibited galactosidase activity significantly augmented over background levels. Effective gene transfer was shown to require both the DNA-binding moiety and cognate moiety for the cell surface receptor. Specific internalization of the complex by the Fc receptor pathway was verified by competitive inhibition using excess IgG. Under this condition, LacZ gene expression was inhibited, suggesting complex internalization through the Fc mediated endocytosis pathway. The requirement of Fc receptors for complex internalization was further demonstrated using cells that lack Fc receptors, e.g., alveolar epithelial cells. When exposed to the IgG-pL/pSVβ complex, these epithelial cells showed no susceptibility to gene transfer. Thus, the immune conjugate system may be used to accomplish targeted gene delivery to the AMs via the endocytosis pathway. Finally, the conjugate system was found to be nontoxic at concentrations effectively enhancing gene transfer, thereby, suggesting its potential safety in vivo.