Differential cell reaction upon Toll-like receptor 4 and 9 activation in human alveolar and lung interstitial macrophages

Background

Investigations on pulmonary macrophages (MΦ) mostly focus on alveolar MΦ (AM) as a well-defined cell population. Characteristics of MΦ in the interstitium, referred to as lung interstitial MΦ (IM), are rather ill-defined. In this study we therefore aimed to elucidate differences between AM and IM obtained from human lung tissue.

Methods

Human AM and IM were isolated from human non-tumor lung tissue from patients undergoing lung resection. Cell morphology was visualized using either light, electron or confocal microscopy. Phagocytic activity was analyzed by flow cytometry as well as confocal microscopy. Surface marker expression was measured by flow cytometry. Toll-like receptor (TLR) expression patterns as well as cytokine expression upon TLR4 or TLR9 stimulation were assessed by real time RT-PCR and cytokine protein production was measured using a fluorescent bead-based immunoassay.

Results

IM were found to be smaller and morphologically more heterogeneous than AM, whereas phagocytic activity was similar in both cell types. HLA-DR expression was markedly higher in IM compared to AM. Although analysis of TLR expression profiles revealed no differences between the two cell populations, AM and IM clearly varied in cell reaction upon activation. Both MΦ populations were markedly activated by LPS as well as DNA isolated from attenuated mycobacterial strains (M. bovis H37Ra and BCG). Whereas AM expressed higher amounts of inflammatory cytokines upon activation, IM were more efficient in producing immunoregulatory cytokines, such as IL10, IL1ra, and IL6.

Conclusion

AM appear to be more effective as a non-specific first line of defence against inhaled pathogens, whereas IM show a more pronounced regulatory function. These dissimilarities should be taken into consideration in future studies on the role of human lung MΦ in the inflammatory response.     

Selective binding and oligomerization of the murine granulocyte colony-stimulating factor receptor by a low molecular weight,nonpeptidyl ligand

Granulocyte colony-stimulating factor regulates neutrophil production by binding to a specific receptor, the granulocyte colony-stimulating factor receptor, expressed on cells of the granulocytic lineage. Recombinant forms of granulocyte colony-stimulating factor are used clinically to treat neutropenias. As part of an effort to develop granulocyte colony-stimulating factor mimics with the potential for oral bioavailability, we previously identified a nonpeptidyl small molecule (SB-247464) that selectively activates murine granulocyte colony-stimulating factor signal transduction pathways and promotes neutrophil formation in vivo. To elucidate the mechanism of action of SB-247464, a series of cell-based and biochemical assays were performed. The activity of SB-247464 is strictly dependent on the presence of zinc ions. Titration microcalorimetry experiments using a soluble murine granulocyte colony-stimulating factor receptor construct show that SB-247464 binds to the extracellular domain of the receptor in a zinc ion-dependent manner. Analytical ultracentrifugation studies demonstrate that SB-247464 induces self-association of the N-terminal three-domain fragment in a manner that is consistent with dimerization. SB-247464 induces internalization of granulocyte colony-stimulating factor receptor on intact cells, consistent with a mechanism involving receptor oligomerization. These data show that small nonpeptidyl compounds are capable of selectively binding and inducing productive oligomerization of cytokine receptors.     

Interleukin-1F7B (IL-1H4/IL-1F7) is processed by caspase-1 and mature IL-1F7B binds to the IL-18 receptor but does not induce IFN-gamma production

We have recently reported the identification of four novel members of the interleukin-1 (IL-1) family which we designated as IL-1 homologue 1-4 (IL-1H1-4). These proteins exhibit significant sequence homology to other members of the IL-1 family. Of these homologues, only IL-1H4 (renamed IL-1F7b) was predicted to contain a propeptide domain and a caspase cleavage site. We now report that caspase-1 cleaves IL-1F7b at the predicted site to generate mature IL-1F7b. Caspase-4 was also able to process IL-1F7b, albeit inefficiently. Other caspases and Granzyme-B did not cleave IL-1F7b. Furthermore, adenovirus-mediated expression of IL-1F7b in HEK 293 cells led to in situ processing and secretion of mature IL-1F7b. In a screen to identify a potential receptor, both pro and mature IL-1F7b bound to the soluble IL-18 receptor alpha-Fc (IL-18Ralpha-Fc) but not to the soluble IL-1R-Fc or ST2R-Fc fusion proteins. Mature IL-1F7b bound to the IL-18Ralpha-Fc protein with higher affinity than the pro form, although the affinities for both proteins were significantly lower than that observed for IL-18. Consistent with this observation, only IL-18 and not IL-1F7b induced IFN-gamma production by KG1a cells. We also report that pro and mature IL-1F7b form homodimers with association constants of 4 microM and 5 nM, respectively, suggesting biological relevance to IL-1F7b processing. Finally, we have localized the expression of IL-1F7b protein in discrete cell populations including plasma cells and tumor cells. These data suggest that IL-1F7b may be involved in immune response, inflammatory diseases and/or cancer.     

Biomonitoring air pollution with the desert lichen Ramalina maciformis

To investigate the environmental impact of anthropogenic activity in the Negev Desert, we monitored the following in order to determine the physiological integrity of the epilithic fruticose lichen Ramalina maciformis: (1) production of ethylene; (2) potential quantum yield of photosystem II expressed as chlorophyll fluorescence ratio Fv/Fm; and (3) electrolyte leakage of cell membranes in terms of electric conductivity. The degree of pollution was assessed by measuring the amounts of B, Cd, Co, Cu, Fe, K, Mg, Mn, Na, Ni, P, Pb, Sr and Zn in the lichen thallus. Some of the lichen-carrying stones collected in the relatively unpolluted control site were relocated on the same hill, to test the possible impact of relocation. An additional amount of lichen-carrying stones was marked as controls. The greater part of the lichen-carrying stones was transferred to 10 biomonitoring sites in and around Ramat Hovav, an industrial area in the Negev Desert, Israel. Transplanted lichen in polluted sites contained higher amounts of mineral elements than lichens in the control site after an exposure period of 7 months. Statistical analysis revealed positive correlation of ethylene production and Ni content, negative correlation of Fv/Fm ratios and B, Cu, Mn, Na, Ni, Sr and Zn, and positive correlation of electric conductivity and B, Mn, Ni and Sr. Both elemental content and physiological alterations in transferred samples of R. maciformis point to a high degree of contamination in Ramat Hovav. The present study suggests that the lichen R. maciformis should be classified as a sensitive species endangered by anthropogenic activity in the desert. Furthermore, this lichen species is likely to be suitable as a bioindicator of pollution in other parts of the Negev Desert. Ethylene production and electric conductivity as parameters of lichen-vitality, indicated stress in 5 of the 12 biomonitoring sites (42%) and may therefore be considered as more sensitive than the Fv/Fm ratio, which indicated stress in 3 of the 12 sites (25%).     

Oral endotracheal intubation of rats for intratracheal instillation and aerosol drug delivery.

As reported in the literature, oral endotracheal intubation of rats is considered to be very difficult. Specialised equipment and complicated techniques have been described to perform this procedure. In our experiment we adopted a simple method, which allowed-without any complicated equipment-the insertion of a relatively wide tube into the trachea of rats, allowing drug administration.     

Development of a new aerosol delivery system for systemic pulmonary delivery in anaesthetized and orotracheal intubated rats.

Over the last decade, the systemic absorption of a broad range of therapeutics after pulmonary application has been demonstrated in animals as well as in humans. The most common method used in the laboratory is the intratracheal instillation of drugs in solution. This method is, however, unsatisfactory, because of discrepancies in particle distribution, clearance, kind of injury and bioavailability between instillation and inhalative application. On the other hand, a precise determination of the amount of drug applied by aerosol, and of the aerosol volume retained within the lungs is rather difficult, and is not possible for use with small animals such as mice or rats. We describe a system which allows the delivery of aerosols directly into the animal's lungs, and calculation of the amount of drug retained in the lungs. Our system was tested in vitro and in vivo and was shown to allow precise and efficient pharmacokinetic and toxicological studies to be carried out.     

Wading bird use of established and newly created reactor cooling reservoirs at the Savannah River Site,near Aiken,South Carolina,USA

We compared wading bird use of a newly created 405-ha reactor cooling reservoir (L-Lake) at the Savannah River Site, near Aiken, South Carolina, with that of two similar > 25-year-old reservoirs (1130-ha Par Pond and 87-ha Pond B) at the same site. L-Lake was constructed in 1984–1985 and filled in late 1985. Approximately 25% of the shoreline of L-Lake was planted with lacustrine vegetation in early 1987 in an attempt to speed the establishment of a self-sustaining balanced biological community (BBC) at the reservoir. Even so, during the course of our studies, L-Lake had considerably less wetland vegetation, especially floating-leaved species such as yellow nelumbo (Nelumbo lutea), than did either of the two older reservoirs.Sixty-three surveys of the avian communities using the three reservoirs were conducted between fall of 1987 and summer of 1989. These surveys indicated that (1) at least seven species of wading birds (order: Ciconiiformes) used L-Lake, (2) wading bird density was higher at L-Lake than at the other two reservoirs, (3) wading birds represented a higher proportion of the total avian community at L-Lake than at the other two, older reservoirs, and (4) wading birds at L-Lake used planted portions of the shoreline more than unplanted portions. We suggest that the inter-reservoir differences we observed represent a trophic upsurge (sensu O'Brien, 1990) resulting from the increased availability of fishes at L-Lake compared with the two older reservoirs.Work was performed at the University of Georgia's Savannah River Ecology Laboratory, Aiken, South Carolina 29802.     

New nanocomposite films for biosensors: layer-by-layer adsorbed films of polyelectrolytes, proteins or DNA

This report describes the construction of ultrathin multicomponent films with an internal structure on the nanometre scale. In the simplest case, the films are built-up by the subsequent adsorption of polyanions and polycations. They can be fabricated on inorganic substrates such as glass, quartz or silicon wafers, or on various organic materials. The polymeric interlayers can incorporate materials with desired electrical or optical properties. The average thickness of the layers can be fine-tuned with Angstrom precision by the addition of suitable salts. They are temperature stable up to at least 200°C and can be laterally structured using conventional photolithographic techniques. The films provide for a well-defined substrate-independent interface for the immobilization of biological macromolecules, such as proteins or DNA, in their active state. The immobilization of streptavidin enables the controlled attachment of any biotinylated molecule with no resulting loss in its biological activity. Area-selective immobilization provides the possibility of built-in quality control for the fabrication of biosensors with separated reference and sample areas on the same substrate.