The inability of CF airway cells to clear P. aeruginosa leads to the
influx of neutrophils into the airways. This inflammatory response appears
coordinated by the epithelial cells. The bacteria trigger the airway epithelia
to secrete proinflammatory cytokines (such as interleukin-8). The bacterial factor(s) responsible for triggering the epithelial inflammatory
response and the signaling pathway(s) activated remain a mystery.
Hypothesis: P. aeruginosa secrete or release factors (e.g.,
lipopolysaccharide, LPS) that bind to receptors (e.g., toll-like receptors)
that in turn activate MAPK and NFKB signaling pathways, resulting in the up- and down-regulation of families of genes
involved in inflammation and facilitating the recruitment and transmigration of neutrophils.
We are measuring gene expression in response to P. aeruginosa using two
methods. In collaboration with Steve Lory (Harvard) we have
been performing gene microarray studies to determine the wide range of
are activated. Airway epithelial cells are grown to confluence, and P.
aeruginosa are added selectively to the apical side. After different
is extracted and gene microarray analysis is performed. Approximately 150 genes (out of about 5000) were activated by
and the patterns of gene expression indicate that the MAP kinase pathway and
several different transcription factors that are commonly associated with
inflammatory reactions are all upregulated. In addition, many genes associated
with epithelial integrity (e.g., junctional proteins) are down regulated while
those associated with neutrophil migration (IL-8, ICAM's and integrins) are
upregulated. These studies will be continued using the Functional Genomics
Center here at UC-Berkeley.
In addition, we are setting up a fluorescence method for measuring P.
aeruginosa-induced IL-8 gene expression in single living airway epithelial
This method has been pioneered by Tsien and his colleagues (__), and we are
using the same approach to determine the time course, cell specificity and the
receptors and second messengers involved in the P. aeruginosa activation of
gene expression. The combined use of the microarray and the fluorescence
will allow us great flexibility in determining the receptors, second messenger
pathways and specific genes activated during the presence of apical P.
in the airways of CF individuals. We will also study any differences in
signalling between CF and wt epithelia.
Epithelial Signaling Pathways Induced by P. aeruginosa
Hypothesis: P. aeruginosa (using the bacterial protein
pilin) bind to
receptors (asialo-GM1) on the epithelial cells that then trigger the second
messenger Ca+2 and upregulation of the transcription factor NFAT
to activate the
Normal and CF airway epithelial cells are grown to confluence on filters,
with the Ca-sensitive dye fura-2 and mounted on the stage of a digital imaging
microscope for measurement of cytosolic [Ca+2]. Our approach
(see Sjaastad et
al, 199_) allows us to recreate the physiological condition and add P.
and also to perfuse different solutions onto the apical and basolateral sides
selectively. We have found that P. aeruginosa bind only to
sites (Lee et al, 2000) of confluent airway epithelia, and that apical bacteria
have no effect on airway epithelial cell [Ca+2]. Basolateral
addition of P.
aeruginosa leads to increases in [Ca+2], but only if the PA
cytotoxic. Use of mutant strains of P.
aeruginosa and different perfusing solutions allowed us to conclude that
cytotoxic (but not noncytotoxic) P. aeruginosa elicit increases in
epithelial Cai by using pili to bind to the basolateral
membranes and then
secreting exotoxin U into the epithelial cells; this causes, after a delay of
30-50 mins, an inhibition of Ca+2 pumps in the endoplasmic
reticulum and activation
of store-activated Ca+2 channels in the basolateral membranes of
cells, leading to increases in cell [Ca+2] that precede, but are
for, cell killing. Because apical P. aeruginosa do not activate airway
epithelial cell [Ca+2], we conclude that in the physiological
aeruginosa binding and Ca+2 signaling is irrelevant for
activating IL-8 or other
genes. We have now begun to investigate the role of Ca+2 in the