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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online March 20, 2001 as doi:10.1096/fj.00-0742fje. |
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Pulmonary, Allergy and Critical Care Division, Department of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA;
* Department of Medicine, Division of Pulmonary Medicine and Critical Care, Jefferson Medical College, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA;
Department of Biochemistry and Molecular Biology, Georgetown University Medical Center, Washington, D.C. 20007, USA; and
Department of Microbiology and Immunology, Kimmel Cancer Institute, Thomas Jefferson University, Philadelphia, Pennsylvania, 19107, USA
2Correspondence: University of Pennsylvania Medical Center, 805 BRB II/III, 421 Curie Blvd., Philadelphia, PA 19104-6160, USA. E-mail: rap{at}mail.med.upenn.edu
SPECIFIC AIMS
Our central hypothesis states that sphingosine 1-phosphate (SPP) is an important inflammatory mediator implicated in the pathogenesis of asthma. We found that SPP levels were elevated in the airways of asthmatic (but not control) subjects after segmental antigen challenge and that, in vitro, SPP modulates human airway smooth muscle (ASM) contraction, cell growth, and proinflammatory cytokine production that promote bronchoconstriction, airway inflammation, and remodeling in asthma.
PRINCIPAL FINDINGS
1. SPP is increased after segmental allergen challenge
(SAC) in asthmatic subjects
Bronchoalveolar lavage (BAL) fluid collected from
asthmatic subjects (n=7) 1 day after SAC possessed a
significantly greater (P<0.05) number of eosinophils,
lymphocytes, and neutrophils than that collected prechallenge. Protein
levels were also markedly elevated in the asthmatic group 1 day after
SAC (P < 0.05). In comparison, BAL fluid obtained after SAC
from control subjects (n=7) showed no significant increases
in cell numbers or protein levels when compared with fluid obtained
prechallenge.
A potential role of SPP as a mediator of airway inflammation was
investigated by quantifying SPP levels in recovered BAL fluid
(Fig. 1
). SPP levels were slightly but not significantly higher in prechallenge
BAL fluid from asthmatic airways when compared with prechallenge levels
in the control group. SAC of asthmatic subjects, however, induced a
significant increase (P<0.05) in SPP levels measured in BAL
fluid obtained 1 day postchallenge, whereas SPP levels in BAL fluid
from controls subjects were unaffected by SAC. SPP levels induced by
SAC in the asthmatic group correlated with the degree of inflammation
reflected in eosinophil number
(r2=0.726: P=0.0149) and
protein influx (r2=0.858:
P=0.0027) recovered in BAL fluid 1 day after challenge.
These findings show that extracellular levels of SPP are significantly
elevated and track with markers of airway inflammation after exposure
to allergen in the asthmatic airways.
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2. Human ASM cells express EDG receptors.
SM plays a central role in the acute and chronic features
of asthma. To explore the potential role of SPP in asthma, we examined
the effects of exogenous SPP on various physiologically relevant
functions of human ASM cultures. SPP is a high-affinity ligand for
G-protein-coupled receptors known as endothelial differentiation
gene-encoded (EDG) receptors. RT-PCR of human ASM mRNA showed that
cultured ASM express EDG receptors 1 through 7.
3. SPP activate signaling pathways that regulate ASM
contraction
We next examined the capacity of SPP to induce hydrolysis
of membrane phosphoinositides (PI) and intracellular calcium
mobilization, the acute signaling events that promote ASM contraction.
SPP stimulated a small increase in PI turnover relative to that
stimulated by thrombin or bradykinin, both effective activators of PI
turnover, Ca2+ flux, and ASM contraction. This
increase in PI turnover was unaffected by pertussis toxin (PTX)
pretreatment, which ADP ribosylates and inactivates
Gi/o proteins, suggesting that activation of
Gi-coupled receptors is not involved in PI
turnover elicited by exogenous SPP. In a similar manner, SPP caused a
small but discernable increase in intracellular
Ca2+ that was also pertussis toxin insensitive.
4. SPP stimulates ASM cell growth
We also examined whether SPP modulated proliferation of
human ASM cells. SPP (0.1–10 µM) caused a dose-dependent increase in
both DNA synthesis and cell proliferation. At 10 µM, SPP induced a
19 ± 2-fold increase in [3H]thymidine
incorporation and a 31 ± 8% in cell number. SPP-mediated
increases in [3H]thymidine incorporation were
eliminated by pertussis toxin pretreatment, suggesting a predominant
role for Gi-protein-coupled receptor activation
in SPP-mediated growth. SPP also augmented DNA synthesis induced by
either EGF or thrombin, known ASM cell mitogens.
SPP appeared to mediate its mitogenic effects by accelerating progression of ASM cells from G1 to S phase of the cell cycle. In addition, SPP affected a critical signaling event linked to cell cycle progression, i.e., the coordinated increase of cyclin D1 expression with titration of the cyclin-dependent kinase inhibitor p27kip-1. Stimulation of human ASM cells with thrombin, EGF, or SPP caused both an increase in cyclin D1 levels and a decrease in p27kip-1. Moreover, SPP enhanced the stimulatory/inhibitory effect of EGF and thrombin on cyclin D1/p27kip-1 expression.
5. SPP regulates the secretion of cytokines in human ASM
cells
Recent evidence suggests that ASM is not only the primary
effector cell regulating bronchomotor tone, but also plays a role in
orchestrating and perpetuating airway inflammation, a hallmark of
asthma. In response to tumor necrosis factor 
(TNF-), ASM
synthesizes both interleukin 6 (IL-6) and RANTES, molecules that
regulate inflammatory cell trafficking in asthmatic airways. Because
TNF- stimulates sphingomyelinase activity and downstream SPP
production, SPP may be an important effector molecule mediating the
actions of these cytokines. We therefore examined whether SPP modulates
ASM RANTES and IL-6 secretion. Treatment of human ASM cells with SPP
alone (for 18 h) stimulated IL-6 secretion (Fig. 2A
). The increase in IL-6 secretion induced by 1 µM SPP
(3.6 ± 0.2 ng/ml) was similar to that induced by 10 ng/ml TNF-
(4.0 ± 0.7 pg/ml). Combined treatment of ASM cells with SPP and
TNF- resulted in a greater than additive effect on IL-6 secretion
(Fig. 2B
). As with the effect of SPP treatment alone, the
potentiating effect of SPP on IL-6 secretion was pertussis toxin
insensitive. In contrast to its observed effects on IL-6 secretion, SPP
significantly inhibited TNF--induced RANTES secretion in
a dose-dependent, pertussis toxin-insensitive manner (Fig. 2C
). Thus, with respect to RANTES secretion, SPP effects are
clearly antithetical to those of TNF-.
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Activation of heterotrimeric G-proteins has varied effects on
human ASM adenylyl cyclase activity and cAMP accumulation that are
related to duration of activation, although the predominant effect of
acute activation is either inhibition (Gi) or no
effect (Gq, G12/13).
However, SPP has been observed to stimulate cAMP accumulation in
certain cell systems, possibly via a Gs-coupled,
EDG receptor-mediated mechanism. To explore the potential role of cAMP
as a mediator of these SPP-dependent effects on cytokines, we first
examined the effects of acute addition of SPP to human ASM cultures on
intracellular cAMP accumulation. SPP caused a significant
dose-dependent, pertussis toxin-insensitive increase in cAMP
accumulation in human ASM cells. Paradoxically, SPP significantly
inhibited, with relatively high-affinity, forskolin-stimulated cAMP
accumulation, and this inhibition was largely pertussis toxin
sensitive. We next examined the effect of chronic treatment with SPP on
adenylyl cyclase responsiveness. Exposure of ASM to 0.1–10 µM SPP
for 18 h increased cAMP formation elicited by stimulation with
forskolin. The increase in intrinsic adenylyl cyclase activity was
inhibited by pertussis toxin, suggesting a
Gi-coupled, EDG receptor-mediated mechanism.
Although the significance of adenylyl cyclase sensitization is unclear,
one possible role may be to preserve the ability of cells to effect
cAMP-mediated signaling important to cell-specific functions, perhaps
by offsetting the inhibitory (and pertussis toxin-sensitive) effect of
Gi activation on
adenylyl cyclase activity. Thus, the sensitization of adenylyl cyclase
and the acute effects of SPP on cAMP accumulation suggest a possible
cAMP-dependent mechanism by which SPP regulates the secretion of
cytokines in ASM.
CONCLUSIONS
In this study, we identify SPP as a potential inflammatory mediator in asthma and show that SPP, via mechanisms that imply activation of EDG receptors, modulates ASM cell functions that are critically important in the pathobiology of asthma.
Our findings identified elevated levels of SPP in BAL fluid recovered from the airways of allergic asthmatic subjects 24 h after SAC. This increase in SPP levels induced by SAC in asthmatic airways suggests that extracellular SPP can serve as effector molecule to regulate resident airway cell function.
Our data suggest that exogenous SPP can regulate a variety of important
functions in human ASM, a critically important effector cell in asthma.
Exogenous SPP increased human ASM cell growth in a pertussis
toxin-sensitive manner. In a similar manner, SPP potentiated growth
stimulation by EGF or thrombin. The induction of IL-6 by SPP, as well
as the modulation of TNF--induced IL-6 and RANTES secretion by SPP,
was pertussis toxin insensitive. Moreover, SPP was able to regulate
numerous signaling events in either a pertussis toxin-sensitive
(p42/p44 MAPK phosphorylation and adenylyl cyclase activity) or
-insensitive (PI turnover, Ca2+ mobilization, and
acute cAMP generation) manner. Whereas the pertussis toxin-sensitive
events are likely mediated via EDG receptors linked to
Gi, the pertussis toxin-insensitive events may
reflect either activation of (non-Gi-coupled)
receptors or intracellular actions of internalized SPP (Fig. 3
).
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SPP has been shown to regulate numerous and diverse cell functions; it appears capable of activating myriad signaling pathways and may contribute to multiple disease states. Our findings suggest that the biology and pathogenesis of asthma may also be affected by SPP.
FOOTNOTES
1 To read the full text of this article, go to
http://www.fasebj.org/cgi/doi/10.1096/fj.00-0742fje ; to cite this
article, use FASEB J. (March 20, 2001)
10.1096/fj.00-0742fje 
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denotes significant difference between control and asthmatic subjects
compared on the same postchallenge day (P < 0.05).
