David L. Haviland

A pure population of lung alveolar epithelial type II cells derived from human embryonic stem cells.

Alveolar epithelial type II (ATII) cells are small, cuboidal cells that constitute ≈60% of the pulmonary alveolar epithelium. These cells are crucial for repair of the injured alveolus by differentiating into alveolar epithelial type I cells. ATII cells derived from human ES (hES) cells are a promising source of cells that could be used therapeutically to treat distal lung diseases. We have developed a reliable transfection and culture procedure, which facilitates, via genetic selection, the differentiation of hES cells into an essentially pure (>99%) population of ATII cells (hES-ATII). Purity, as well as biological features and morphological characteristics of normal ATII cells, was demonstrated for the hES-ATII cells, including lamellar body formation, expression of surfactant proteins A, B, and C, α-1-antitrypsin, and the cystic fibrosis transmembrane conductance receptor, as well as the synthesis and secretion of complement proteins C3 and C5. Collectively, these data document the successful generation of a pure population of ATII cells derived from hES cells, providing a practical source of ATII cells to explore in disease models their potential in the regeneration and repair of the injured alveolus and in the therapeutic treatment of genetic diseases affecting the lung.

Expression of the receptors for the C5a anaphylatoxin, interleukin-8 and FMLP by human astrocytes and microglia

The expression of chemotactic receptors in the central nervous system is largely unexplored. In this study, we examined human astrocytes and microglia as well as the conditionally immortalized human astrocyte cell line HSC2 for expression of the C5a-anaphylatoxin receptor (C5aR), the interleukin-8 receptor (IL-8R) and the f-Met-Leu-Phe receptor (FMLPR). Using flow cytometry, indirect immunofluorescence and RT-PCR analysis, we demonstrated that astrocytes, microglia and HSC2 cells contain specific RNA and express surface protein for all three chemotactic receptors. These are the first studies to demonstrate definitively the expression of these chemotactic receptors astrocytes and microglia, thereby expanding the types of cells known to express chemotactic receptors. Moreover, these data suggest that these chemotactic receptors may play an important role in mediating the inflammatory response and perhaps other yet undescribed biological phenomena in the central nervous system.

Inhibition of Complement Activation by a Secreted Staphylococcus aureus Protein

Staphylococcus aureus can cause a variety of acute and chronic diseases. The ability of S. aureus to cause persistent infections has been linked to its ability to evade or inactivate host immune responses. We have identified a secreted 19-kDa protein produced by S. aureus that binds to the complement protein C3. N-terminal sequencing of this protein identified it as the extracellular fibrinogen-binding protein (Efb). In this study, we demonstrate that Efb can bind to the alpha -chain of C3 and inhibit both the classical and alternative pathways of complement activation. In addition, we show that Efb can inhibit complement-mediated opsonophagocytosis in a dose-dependent manner and that Efb inhibits complement activity by blocking deposition of C3 or by preventing further complement activation beyond C3b. These data suggest that Efb is a virulence factor involved in facilitating persistent S. aureus infections by interfering with complement activity in vivo.

Identification, Cloning, and Functional Characterization of a Murine Lipoxin A4 Receptor Homologue Gene

To identify additional members of the murine N-formyl-Met-Leu-Phe peptide receptor family (fMLF-R), a mouse macrophage cDNA library was screened using the open reading frame of murine N-formyl peptide receptor. Four individual hybridizing cDNA clones were maintained through tertiary screening. One cDNA clone was a truncated, polyadenylated version of the previously described murine-fMLF-R. The other three cDNA clones varied in length, but contained identical open reading frame sequences. One clone, 8C10, was selected for further study and shared 70% sequence identity with murine-fMLF-R and 89% sequence identity with murine lipoxin A4 receptor cDNA. When placed into the pcDNA-3 expression vector and cotransfected with Gα16 cDNA into COS-1 cells, 8C10 cDNA induced the production of inositol-1,4,5-triphosphate when concentrations of 1–1600 nM lipoxin A4 (LXA4) were tested as ligands. Northern blot analysis of murine organs indicated that the 8C10 message is present in lung, spleen, and adipose tissue. Moreover, mice treated with LPS demonstrated increased expression of 8C10 message in spleen and adipose tissue, while showing a slight reduction in lung. We have also characterized the 8C10 structural gene from a 129Sv/J genomic library and have determined its size to be >6.1 kb in length and comprised of two exons separated by a 4.8-kb intron. Collectively, these data indicate that this homologue receptor is closely related to the murine LXA4 receptor and functionally responds to LXA4 as a ligand.

Complement Anaphylatoxins (C3a, C4a, C5a) and Their Receptors (C3aR, C5aR/CD88) as Therapeutic Targets in Inflammation

One of the major biological consequences of complement activation is the generation of three small cationic peptides C3a, C4a, and C5a collectively referred to as complement anaphylatoxins. The word anaphylatoxin was first used by Friedberger to describe the activity found in complement-activated serum, which produced rapid death when injected into laboratory animals (1). It has remained the generic name given to these peptides. As a group, the complement anaphylatoxins are recognized for their potent proinflammatory functions in mediating smooth muscle contraction, histamine release from mast cells, and increased vascular permeability (2–6). In addition, one of the peptides, C5a, is recognized as one of the most potent molecular chemoattractants for neutrophils and other leukocytes (7). The other two peptides, C3a and C4a, were considered for a time not to exhibit any chemoattractant properties; however, during the past few years it has become clear that C3a, although not chemoattractant for neutrophils (8), can cause the directed migration of eosinophils (8), and possibly mast cells (9,10). Presently, it is unclear if C4a is also a chemoattractant molecule.

Complement C5a anaphylatoxin is an innate determinant of dendritic cell-induced Th1 immunity to Mycobacterium bovis BCG infection in mice

During acquired immunity to Mycobacterium bovis bacillus Calmette‐Guerin (BCG) infection in mice, dendritic cells (DCs) present mycobacterial antigens to naive T cells to prime an immune response. Complement C5a (anaphylatoxin) secreted by mycobacteria‐infected macrophages regulates IL‐12p70 production. As IL‐12p70 regulates Th1 immunity against mycobacteria in mice, we examined the effects of C5a on IL‐12p70 secretion by murine DCs and Th1 immunity. DCs cultured from C5‐deficient (C5−/−) and ‐sufficient (C5+/+) mice were infected with BCG in the presence or absence of the C5a peptide. ELISA showed that C5−/− DCs secreted less IL‐12p70 (600 pg/mL vs. 100 pg/mL) than C5+/+ DCs, and they secreted more IL‐10. Using immunophenotyping, reduced CD40 expression was found on C5−/− DCs after BCG infection. BCG‐primed DCs were then cocultured with naive or BCG‐immune T cells to differentiate them into IFN‐γ‐secreting Th1 T cells. Coincident with increased IL‐12p70 levels, BCG‐primed C5+/+ DCs cocultured with naive or immune C5+/+ T cells showed a larger increase in CD4+ IFN‐γ/CD8+ IFN‐γ+ T cells compared with cocultured DCs and T cells from C5−/− mice. Thus, BCG‐primed C5+/+ DCs were better able to drive a Th1 response. Furthermore, BCG aerosol‐infected C5−/− mice showed reduced CD4 and CD8 IFN‐γ‐secreting T cells in the lungs, concurrent with an increased growth of BCG. Thus, C5a, an innate peptide, appears to play an important role in the generation of acquired immune responses in mice by regulating the Th1 response through modulation of IL‐12p70 secretion from DCs.