Please select papers that best represent your most significant work (criteria
would include impact on practice, citation index, impact factor of journals
in which they were published, thematic link to other work you have done).
Suggestion - appointment on the Investigation Area of Expertise would
typically only include peer-reviewed research, while Clinician Expertise &
Innovation might pick 1-3 chapters or reviews in addition to their original
research. Usually the summary is 3-5 sentences about the subject of the
work, the importance of the work, your contribution to the work, and the way
it launched other work or built on prior work. Successful studies with
mentees, papers that won awards at meetings, or work that has otherwise had
an impact would be worth choosing.
If sending a middle author paper, please provide your particular
contribution to this publication, which is what the P&R needs to see when
candidates select middle author publications for submission as part of their
promotion packets. Please explain your role on this study (e.g. design,
research, provided reagents, provided expertise, writing)?
What the P&R committee most likes to see is evidence that the candidate made
an intellectual contribution toward the project and/or provided expertise in
an area that the first/senior authors lacked (e.g. as the statistician for
the group).
Annotated Bibliography
1. Owen, C.A., Campbell M.A., Sannes P.L., Boukedes S.S., Campbell E.J.
Cell surface-bound human leukocyte elastase and cathepsin G on human neutrophils: a novel
non-oxidative mechanism by which neutrophils focus and preserve catalytic activity of serine
proteinases. Journal of Cell Biology 1995;131:775-789.
Background: Neutrophils store preformed neutrophil elastase (NE) and cathepsin G (CG) in their
azurophil granules and release these enzymes into the extracellular space when activated. Extracellular
NE and CG play critical roles in extracellular proteolysis and cause tissue destruction during inflammatory
responses to injury. Prior to publication of this paper, inhibitor-resistant proteolysis was known to be
associated with activated inflammatory, but the mechanisms by which inflammatory cell-derived enzymes
circumvented the high concentrations of proteinase inhibitors present in the extracellular space was not
known. In this manuscript, we were the first to identify a critically important mechanism by which this
occurs: binding of serine proteinases in catalytically active but inhibitor resistant forms on to the surface of
activated neutrophils.
Findings: We showed that when neutrophils are activated with pro-inflammatory mediators, NE and CG
rapidly translocate from the azurophil granules and bind to the external surface of the neutrophil plasma
membrane at sites where the azurophil granules fuse with the plasma membrane. Activated neutrophils
express 20-fold more surface NE and CG than unstimulated neutrophils, and 6-fold more is detected on
the surface compared to the amounts of enzymes that are freely released from the cells. Moreover,
surface-bound NE and CG are catalytically active and can degrade extracellular matrix proteins as
efficiently as the soluble forms of the proteinases. However, membrane-bound NE and CG differ from the
soluble forms of the proteinases by their striking resistance to inhibition by physiologic serine proteinase
inhibitors.
Significance: The binding of NE and CG to the surface of neutrophil serves to focus, preserve and
restrict their biologic activities to the pericellular environment of neutrophils. Also, membrane-bound NE
and CG on activated neutrophils are critically important bioactive forms of the enzymes which are well
equipped to contribute to physiologic and pathologic processes. These results have implications for the
development of new treatments strategies for common diseases in various organ systems in which serine
proteinase-mediated tissue injury is a critical event.
2. Campbell, E.J., Campbell M.A., Boukedes S.S., Owen C.A. Quantum proteolysis by neutrophils:
implications for therapy in α1-antitrypsin deficiency. Journal of Clinical Investigation
1999;104:337-344.
Background: This manuscript reported a second critically important mechanism leading to inhibitorresistant proteolysis associated with activated inflammatory cells (see above): quantum proteolysis. In
addition, this manuscript provided insights into why patients with severe deficiency of alpha-1-antitrypsin
(AAT) due to inheritance of PiZZ alleles develop early onset and very severe pulmonary emphysema
whereas heterozygous individuals (PiMZ or PiSZ) who have ~50% reductions in serum AAT levels do not
have an increased risk of developing pulmonary emphysema.
Findings: Neutrophils store neutrophil elastase (NE) at millimolar concentrations in their azurophil
granules. The release of single azurophil granules of activated neutrophils leads to evanescent quantum
bursts of proteolytic activity (quantum proteolysis) before the enzyme diffuses from the site of
degranulation, its concentration decreases and is quenched by pericellular inhibitors especially AAT in the
extracellular space. We tested the hypothesis that quantum proteolytic events associated with activated
neutrophils are abnormally large in patients with AAT deficiency, but not largely increased in PiMZ and
PiSZ heterozygotes to explain their differences in the risk for developing pulmonary emphysema. We
incubated neutrophils on opsonized and fluoresceinated fibronectin bathed in serum from individuals with
various AAT phenotypes, and then measured and modeled quantum proteolytic events. The mean areas
of the quantum proteolytic events in serum from heterozygous individuals (Pi MZ and Pi SZ) were only
slightly larger than those in serum from subjects with normal serum levels of AAT (Pi MM). In marked
contrast, the mean areas of these events in serum from AAT-deficient individuals were 10-fold larger than
those in serum from normal subjects. Mathematical diffusion modeling predicted that local elastase
concentrations exceed AAT concentrations for less than 20 milliseconds and for more than 80
milliseconds in Pi M and Pi Z individuals, respectively.
Significance: Thus, quantum proteolytic events are abnormally large and prolonged in AAT deficiency,
leading directly to an increased risk of tissue injury in the immediate vicinity of activated neutrophils.
These results have potentially important implications for the pathogenesis and prevention of lung disease
in AAT deficiency. Strategies to increase AAT serum levels to those occurring in heterozygous individuals
likely will be sufficient to reduce lung destruction on PiZZ individuals.
3. Owen, C.A., Hu, Z. Barrick, B., Shapiro, S.D. Inducible expression of TIMP-resistant matrix
metalloproteinase-9 (MMP-9) on the cell surface of neutrophils. Am. J. Resp. Cell and Mol. Biol.
2003; 29:283-294.
Background: Prior to publication of this paper, matrix metalloproteinases (MMPs) were thought to
function exclusively as soluble proteinases released from cells. This manuscript was the first to report
that a matrix metalloproteinase lacking a transmembrane domain (MMP-9, or gelatinase B) is expressed
on a cell surface where it contributes substantially to pericellular, inhibitor-resistant proteolysis in
physiologic and pathologic processes of neutrophils.
Findings: We showed pro-inflammatory mediators that promote neutrophil degranulation, also promote
translocation of MMP-9 from its storage site in intracellular gelatinase granules to the plasma membrane
of activated neutrophils. Activated PMN express large quantities of MMP-9 on their cell surface.
Membrane-bound MMP-9 is catalytically active and can degrade extracellular matrix components (elastin
and denatured collagens) and cleave and inactivate serine proteinase inhibitors with similar catalytic
efficiency as the soluble form of the proteinase. In contrast, to soluble MMP-9, membrane-bound MMP-9
is resistant to inhibition by tissue inhibitors of metalloproteinases (TIMPs) likely via a steric hindrance
mechanism.
Significance: We identified a new bioactive form of MMP-9 which is likely to be the most important
extracellular form of the enzyme contributing to tissue injury. The results of this study have implications for
the development of new treatments strategies for common diseases in various organ systems in which
MMP-9-mediated tissue injury is a critical event. In particular, we showed that unlike large TIMPs, small
synthetic inhibitors are very effective against membrane-bound MMP-9, indicating that synthetic inhibitors
may have therapeutic potential for diseases in which MMP-9-mediated tissue injury is a critically important
event.
4. Owen, C.A., Hu, Z., Lopez-Otin, C., Shapiro, S.D. Membrane-bound matrix metalloproteinase-8
on activated polymorphonuclear cells is a potent, tissue inhibitor of metalloproteinase-resistant
collagenase and serpinase. Journal of Immunology 2004; 172:7791-7803.
Background: Prior to publication of this paper, little was known about the cell biology of MMP-8
(neutrophil collagenase) or its roles in the lung. The main role of MMP-8 was thought to be restricted to
degradation of interstitial collagens because of its substrate specificity is mainly limited to interstitial
collagens when purified MMP-8 is tested in vitro. In this paper, we identified a novel form of MMP-8 in
neutrophils (the major source of this enzyme), which likely is the major bioactive form of this enzyme
expressed when cells migrate into tissues. We also identified for this first time that MMP-8 has a
surprising anti-inflammatory role during LPS-mediated acute lung injury in mice.
Findings: We showed that when neutrophils are activated with pro-inflammatory mediators, or induced to
migrate in response to a chemokine gradient, MMP-8 rapidly translocates from its storage sites in the
specific granules of neutrophils to the plasma membrane. Membrane-bound MMP-8 is catalytically active,
but in contrast to MMP-8 released from the cells, it is resistant to inhibition by TIMPs. In vitro studies of
activated neutrophils from wild type (WT) mice and mice genetically deficient in MMP-8 (MMP8-/-mice)
confirmed that membrane-bound MMP-8 contributes ~100% to the potent pericellular type I collagendegrading activities associated with activated neutrophils. However, when the function of MMP-8 was
assessed in the lung by comparing WT and MMP8-/- mice in a murine model of acute lung injury,
surprisingly, the main role of MMP-8 was not in degrading lung interstitial collagens to promote vascular
leak. Rather that MMP-8 dampens lung inflammation likely by inactivating neutrophil chemokines.
Significance: These results have identified a novel bioactive form of MMP-8 in neutrophils, and a novel
role for this enzyme in the lung. Strategies to increase MMP-8 expression, or half life in the lung may limit
tissue injury in lung diseases associated with intense neutrophilic infiltration.
5. Campbell E.J. and Owen, C.A. Chondroitin sulfate- and heparan sulfate proteolglycans in
neutrophil plasma membranes are novel binding sites for neutrophil elastase and cathepsin G. J.
Biol. Chem. 2007, May 11;282(19):14645-54.
Background: Prior to publication of this paper, the mechanisms by which serine proteinases that lack
transmembrane domains [neutrophil elastase (NE) and cathepsin G (CG) bind to the surface of
inflammatory cells was not known. In addition, the mechanism underlying the resistance of serine
proteinases to inhibition by large, physiologic extracellular inhibitors was thought to be a steric hindrance
phenomenon based upon the indirect relationship between inhibitor size and its effectiveness against
membrane-bound serine proteinases (large physiologic inhibitors are ineffective inhibitors of membranebound serine proteinases, but small, synthetic inhibitors efficiently inhibit membrane-bound NE and CG).
Findings: In this paper, we identified the receptors for NE and CG on the surface of neutrophil, and in
doing so identified the mechanism by which surface-bound serine proteinases are resistant to inhibition by
serine proteinase inhibitors. We used Scatchard analyses, glycanase treatments and immunofluorescence staining and confocal analyses of neutrophils to show that NE and CG bind via their basic
residues to the negatively charged sulfate groups in heparan sulfate- and chondroitin sulfate-containing
proteoglycans (HSPG and CSPG) on neutrophil cell surfaces. HSPG and CSPG are low affinity, high
volume neutrophil surface binding sites for HLE and CG (KD ~10-7M, and ~107 sites/cell) which are well
suited to bind the high concentrations of active serine proteinases released from degranulating
neutrophils. This process restricts the activities of these potent enzymes to the pericellular environment
of neutrophils. Moreover, it is the binding of serine proteinases to HSPG and CSPG that renders these
enzymes resistant to inhibitors of serine proteinases which are present in the extracellular space.
Significance: HSPG and CSPG might represent new therapeutic targets for diseases in which serine
proteinase-mediated tissue injury is a critically important event.