Serine Protease Cathepsin G Regulates Adhesion-Dependent Neutrophil Effector Functions by Modulating Integrin Clustering

Home , Cathepsin G, Protease, Serine protease

View metadata, citation and similar papers at core.ac.uk brought to you by CORE

provided by Elsevier - Publisher Connector

Immunity, Vol. 22, 679–691, June 7, 2005, Copyright ©2005 by Elsevier Inc. DOI 10.1016/j.immuni.2005.03.015 Serine Protease Cathepsin G Regulates Adhesion-Dependent Neutrophil Effector Functions by Modulating Integrin Clustering

Sofia Z. Raptis,1,2 Steven D. Shapiro,3 mediators (Ravetch, 1994). It has been known that IC Pamela M. Simmons,1 Alec M. Cheng,1,2,4 activation of PMNs requires the cooperation of FcγR 1,2, and Christine T.N. Pham * and β2 integrins, particularly Mac-1 (CD11b/CD18) on 1Division of Rheumatology PMNs (Jones et al., 1998; Zhou and Brown, 1994). In Washington University School of Medicine addition, Mac-1-deficient PMNs have blunted re- Saint Louis, Missouri 63110 sponses to IC stimulation in vitro and decreased PMN 2 Department of Pathology and Immunology recruitment in a model of IC-mediated glomerulone- Washington University School of Medicine phritis in vivo (Tang et al., 1997). Thus FcγR and inte- Saint Louis, Missouri 63110 grins play an important role in the development of IC- 3 Department of Medicine mediated inflammation. Pulmonary and Critical Care Cathepsin G (CG), neutrophil elastase (NE), and pro- Brigham and Women’s Hospital teinase 3 (PR3) are a family of highly related neutral Harvard School of Medicine serine proteases expressed in the primary granules of Boston, Massachusetts 02115 PMNs (Owen and Campbell, 1999). The importance of NE and CG during infections has been clearly demonstrated in serine protease-deficient mice. These mice Summary are more susceptible to various pathogens, including gram-positive and -negative bacteria and certain fungal The polymorphonuclear leukocyte (PMN)-derived ser- organisms (Belaaouaj et al., 1998; Reeves et al., 2002; ine proteases play a key role in immune complex (IC)- Tkalcevic et al., 2000). Recent studies have also shown mediated inflammation. However, the mechanisms by that mice lacking in CG and NE are resistant to IC- which these proteases regulate inflammatory re- mediated diseases, further suggesting an active role for sponse remain largely undefined. Here, we show that these proteases in immune responses other than their IC-activated cathepsin G- and neutrophil elastase- antibacterial functions. Consistent with this emerging deficient (CG/NE) PMNs adhered normally to IC- view of protease functions, studies have demonstrated coated surfaces but did not undergo CD11b cluster- that NE-deficient mice are resistant to an experimental ing and failed to initiate cytoskeletal reorganization mouse model of bullous pemphigoid (Liu et al., 2000), and cell spreading. As a result, CG/NE-deficient PMNs and CG/NE mice were resistant to an IC-mediated, neu- exhibited severe defects in MIP-2 secretion and trophil-dependent arthritis model (Adkison et al., 2002). reactive oxygen intermediates production. Exoge- In the later study, there was no PMN accumulation in the joints of CG/NE mice that received arthrogenic anti- nously added CG, but not proteolytically inactive CG, bodies. Currently, the role of serine proteases in PMN was sufficient to restore these defects. These find- transmigration and recruitment remains controversial. ings identify an important role for CG in integrin- Although some studies suggest that serine proteases dependent PMN effector functions that are separate might be required for the migration of PMNs to inflam- from and downstream of integrin-dependent ad- matory sites (Carden and Korthuis, 1996; Carney et al., hesion. 1998; Woodman et al., 1993), others reported that CG and NE are not essential for normal chemotaxis to vari- Introduction ous inflammatory stimuli (Hirche et al., 2004; Huber and Weiss, 1989; Mackarel et al., 1999). Thus, the fact that PMNs are an essential component of the host innate PMNs deficient in CG and NE exhibit normal chemo- response against invading pathogens. However, exces- taxis but do not accumulate in vivo in response to IC- sive PMN responses are thought to contribute to the induced inflammation points to an undefined role for development and perpetuation of autoimmune dis- these proteases in regulating leukocyte recruitment. eases. Although many aspects of PMN activation lead- According to the current paradigm, CG and NE, ing to transmigration across the endothelial barrier secreted upon PMN activation, are thought to degrade have been elucidated, the mechanisms that regulate specific target molecules of the ECM, thus contributing PMN activation and effector functions in the extracellu- to tissue damage and the propagation of inflammation. lar matrix (ECM) are still undefined. Recent reports suggest, however, that serine proteases One of the major mechanisms by which PMNs accu- remain bound to the cell surface membrane upon re- mulate and become activated at sites of inflammation lease from primary granules, where they become resis- in noninfectious diseases is through the interaction with tant to circulating endogenous protease inhibitors pres- IC. Binding of PMNs to IC through their Fc gamma re- ent in the serum (Owen et al., 1995a, 1995b). Hence, ceptors (FcγR) leads to phagocytosis, generation of re- the mode of action of membrane bound serine prote- active oxygen intermediates (ROI), release of proteo- ases may be more complex than previously thought. lytic enzymes, and secretion of soluble inflammatory These studies and the results from the experimental arthritis model aforementioned led us to consider the un- *Correspondence: [email protected] explored notion that the membrane bound form of ser- 4 Present address: Genentech, Inc., South San Francisco, CA ine proteases may directly regulate PMN activation and 94108. effector functions. In this report, we test this hypothesis Immunity 680

1F). In this case, the number of leukocytes recruited at four hr after injection of zymosan was equivalent in both wild-type (wt) and mutant mice (Figure 1D). Zymo- san has been shown to be a strong activator of the complement system, resulting in the formation of C5a, a potent PMN chemoattractant (Fearon and Austen, 1977). Thus, the C5a gradient generated by zymosan is likely responsible for the equivalent numbers of PMNs initially recruited into the air pouch, as CG/NE PMNs chemotax normally in response to C5a (see Figure S1 in the Supplemental Data available with this article online). As C5a has a short half-life, other chemoattrac- tants are likely required to sustain the recruitment of leukocytes over time. In support of this notion, we previously detected a significant reduction in the number of PMNs recruited to the air pouch of CG/NE mice at 12 hr post zymosan injection (Adkison et al., 2002). PMNs themselves have been shown to synthesize and release CXC chemokines when activated (Scapini et al., 2000). PMNs are among the earliest cell types recruited to an inflammatory site, and their ability to secrete chemokines may constitute a mechanism by which PMNs can Figure 1. Defective Chemokine Production in CG/NE Mice influence the early trafficking of inflammatory cells. Be- Air pouches were formed as described in Experimental Procedures. cause CG and NE are PMN-specific proteases, we pos- For IC formation (A–C), mice were injected with 20 mg/kg chicken tulate that these enzymes may directly regulate PMN egg albumin (OVA) intravenously followed by injection of 800 ␮g effector functions that in turn shape the inflammatory of rabbit anti-OVA Abs in the air pouch (n = 4–6 mice per group). responses. Alternatively, mice were injected with 300 ␮g of zymosan in 1 ml of saline in the air pouch (D–F; n = 4 mice per group). Leukocyte counts in air pouch were enumerated after 4 hr; MIP-2 and KC in CG and NE Are Required for Optimal Adhesion- the lavage fluid were measured by ELISA. Asterisks denote signifi- Dependent Effector Functions cant difference between wt and CG/NE mice. *p < 0.05, **p < 0.01, Sustained PMN effector functions in response to IC ***p < 0.001. All values represent mean ± SD. stimulation require adhesion and have been shown to

depend on the ligation of both FcγR and β2 integrins (Jones et al., 1998; Zhou and Brown, 1994). To recapitu- by examining the response of purified PMNs to IC stim- late these requirements in an in vitro system, wt and ulation in an in vitro cell culture system in which PMN CG/NE PMNs were stimulated on immobilized IC in the activation can be evaluated independent of other cell presence of Ca2+ and Mg2+ and TNF-α priming. IC-acti- types. Our findings suggest that neutrophil-derived ser- vated wt PMNs in adherent culture released significant ine proteases regulate PMN effector functions by mod- levels of the chemokine MIP-2, but not KC, IL-1β,or ulating integrin clustering. TNF-α (data not shown). In contrast, MIP-2 release by CG/NE and CG-deficient PMNs was markedly reduced Results compared to wt PMNs (Figure 2A). PMNs lacking NE alone were minimally compromised in MIP-2 release, Defective Chemokine Release in the Subcutaneous suggesting that CG has a critical role in regulating this Air Pouch of CG/NE Mice effector function (Figure 2A). We also examined NADPH The migration of PMNs to inflammatory sites is regu- oxidase activation in response to IC stimulation. Wt lated by the activity of CXC chemokines such as MIP-2 PMNs generated a robust respiratory burst, whereas and KC (Olson and Ley, 2002). We reasoned that the the amount of ROI produced by CG/NE PMNs was re- absence of PMNs in CG/NE joints in the arthritis model duced by >50%. PMNs lacking CG exhibited a more was due to a defect in the local generation of a chemo- severe defect than those lacking NE, suggesting that tactic gradient. To test this idea, we turned to the sub- CG has a predominant role in IC-mediated ROI pro- cutaneous air pouch model that offered us a direct way duction (Figure 2B). Taken together, these results sug- to assess the number of leukocytes recruited and the gest an unexpected role for PMN-derived serine prote- chemokine response to specific stimuli. When inflam- ases—that of modulating PMN effector functions during mation was induced by in situ IC formation and air adhesion-dependent IC stimulation—as demonstrated pouches were enumerated for leukocyte counts, CG/ by the release of chemokines and ROI production. NE mice exhibited a significant reduction in cell number Moreover, this regulatory effect is mediated mainly by (>50%, p < 0.05) (Figure 1A) as well as chemokine pro- CG, indicating that CG and NE are not absolutely reduction (>3-fold reduction in MIP-2 and KC, p < 0.01) dundant in function. (Figures 1B and 1C). Air-pouch inflammation induced To test if the reduction in MIP-2 secretion and ROI by the yeast particle zymosan also led to a significant production was the result of intrinsic defects in CG/NE reduction in MIP-2 and KC release (p < 0.01 and 0.001, PMNs, we examined PMN effector functions in re- respectively) in the CG/NE air pouch (Figures 1E and sponse to phorbol 12-myristate 13-acetate (PMA), a Cathepsin G Regulates Neutrophil Functions 681

Figure 2. Defective Adhesion-Dependent Functions in CG/NE PMNs MIP-2 release (A) and ROI production (B) by TNF-α-primed, IC-stimulated PMNs were measured over time. MIP-2 release at 2 hr (C) and ROI production (D) by wt and protease-deficient PMNs stimulated with PMA. Data represent the mean ± SD of duplicate samples for MIP-2 and triplicate samples for ROI and are representative of at least four independent experiments. Data are presented as percentage of ROI produced by wt PMNs at 150 min. (E) PMNs were stimulated on anti-OVA Abs or IC in the presence of brefeldin A and stained for intracellular MIP-2. (F) The percentage of MIP-2 released was calculated by dividing MIP-2 in the supernatant by the total amount of MIP-2. Data are representative of four independent experiments. pharmacologic agent known to bypass adhesion-depen- sis or decreased release of the chemokine. Wt and CG/ dent activation of PMNs. Both MIP-2 and ROI pro- NE PMNs produced similar levels of MIP-2 by intracel- duction were comparable in wt and protease-deficient lular staining (Figure 2E). Moreover, detection of MIP-2 PMNs (Figures 2C and 2D), suggesting that these en- transcripts by reverse-transcriptase-polymerase chain zymes are not required when surface receptor ligation reaction (RT-PCR) from IC-stimulated PMNs revealed is bypassed. In contrast, MIP-2 release was not de- no significant difference between wt and CG/NE cells tected when cells were stimulated in suspension with (data not shown). In contrast, wt PMNs released ap- other PMN agonists such as TNF-α or formyl-methio- proximately 46% of the total MIP-2 during IC stimula- nyl-leucyl-phenylalanine (Figure S2). tion, whereas CG/NE PMNs released less than 15% of the total MIP-2 (n = 4, p < 0.001) (Figure 2F). Studies CG and NE Are Required to Regulate MIP-2 Release have shown that membrane shedding of CXC chemo- We next determined if reduced MIP-2 levels by IC-stim- kine by proteases regulates their bioavailability (Li et ulated CG/NE PMNs were a result of decreased synthe- al., 2002). To rule out the possibility that MIP-2 re- Immunity 682

mained bound on the cell surface, we stained nonper- PMN-Derived Serine Proteases Regulate meabilized PMNs for MIP-2 with specific monoclonal Cytoskeleton Reorganization and Integrin Clustering Abs but did not detect membrane expression of the Because adhesion has been linked to chemokine ex- chemokine on activated PMNs by flow cytometry (data pression and shown to influence ROI production in not shown). Together, these results suggest that the de- PMNs (Kessel et al., 1998; Rezzonico et al., 2001; Shap- crease in MIP-2 levels by CG/NE PMNs is due to a fail- pell et al., 1990), we next determined whether activated ure in release and not a failure in synthesis or mem- CG/NE PMNs adhered normally on immobilized IC. We brane shedding of MIP-2. observed no significant difference in the number of ad- We next determined if release of prestored granule herent CG/NE PMNs on IC-coated surfaces (Figure 4A). content was also affected in CG/NE PMNs. Secretory In addition to adhesion, a hallmark response of acti- granule release, as measured by the upregulation of vated PMNs is the initiation of cytoskeletal reorganiza- CD11b in the presence of TNF-α, was normal in prote- tion and cell spreading after adhesion (Yamada and Mi- ase-deficient PMNs compared with wt PMNs (Figure yamoto, 1995). To investigate if CG and NE are required S3A). Likewise, release of MMP-9 from gelatinase (terti- to regulate this response, we assessed F-actin poly- ary) granules and β-glucuronidase from primary gran- merization and cell spreading with phalloidin staining ules was equivalent in all genotypes (Figures S3B and and fluorescence microscopy. In contrast to wt PMNs, S3C). These results suggest that release of prestored CG/NE PMNs exhibited significantly reduced cell spread- granule content proceeds normally in the absence of ing and failed to form a distinct lamellipodia that was serine proteases. found in activated wt PMNs (Figure 4B, top). CXC chemokines have been shown to trigger in- Because cytoskeletal reorganization is critically linked crease in integrin affinity leading to enhanced integrin- to signals from integrins (DeMali et al., 2003), the lack mediated adhesive functions (Laudanna et al., 2002). of cell spreading seen in CG/NE PMNs suggests a defect in integrin-dependent functions. Previous studies of Because the receptor for MIP-2 (the G protein-coupled integrin signaling identified an inside-out signaling step receptor CXCR2) is present on PMNs, MIP-2 could po- initiated from specific receptors (i.e., FcγR ligation in tentially enhance MIP-2 release and ROI production by PMNs) that is first required to induce conformational binding to CXCR2. To examine whether released MIP-2 changes in integrins, leading to increased receptor af- activates integrin-dependent functions in this in vitro finity for ligand binding. This initial activation step re- system, we pretreated wt and CG/NE PMNs with dif- leases integrins from the actin cytoskeleton restraint, ferent doses of pertussis toxin (PTX), an inhibitor of G allowing lateral mobility of integrins within the plasma protein-coupled receptors (Baggiolini et al., 1997). PTX membrane, leading to increased integrin avidity and en- did not affect MIP-2 release by wt or CG/NE PMNs (Fig- hancing subsequent cellular responses (outside-in sig- ure S4A) but blocked chemotaxis induced by IL-8 (the naling). In many cell systems, this increase in avidity is homologue of MIP-2, Figure S4B). In addition, PMNs appreciated in the form of receptor clustering, in which deficient in CXCR2 released equivalent levels of MIP-2 activated integrins appear as distinct aggregates that and ROI compared to wt PMNs stimulated with IC (Fig- can be detected by immunofluorescence microscopy ures S4C and S4D). These results suggest that released (Hogg et al., 2002). Because CD11b is essential for the MIP-2 does not enhance adhesion-dependent PMN ef- activation of PMNs on IC, we examined the role of CG/ fector functions in this in vitro system. NE in regulating the clustering pattern of CD11b. IC- activated, adherent wt PMNs displayed a distinct circu- lar pattern of CD11b staining, probably representing CG and NE Are Required for Optimal PMN points of contact with IC-coated surfaces (Figure 4B, Activation on Integrin Ligands top). These CD11b aggregates were essentially absent We next examined the requirement for serine proteases in CG/NE PMNs (Figure 4B, middle). This difference in when PMNs were directly plated on surfaces coated appearance was not the result of a lower density of β2 β β with ligands for both 1 and 2 integrins. ROI prod- integrins on CG/NE PMNs, as flow cytometry revealed β uction on both fibrinogen (a ligand for 2 integrin) and similar surface expression of CD11b between wt and β fibronectin (a ligand for 1 integrin) was reduced in CG/ CG/NE PMNs (Figure 4C). Hence, CG and NE have a NE PMNs (Figures 3A and 3B). Next we examined critical role in the regulation of integrin clustering in ac- MIP-2 release by PMNs plated on these ligands. Wt and tivated PMNs. CG/NE PMNs plated on fibrinogen and fibronectin did not release any detectable amount of MIP-2, even in CG and NE Are Required to Regulate Intracellular the presence of TNF-α priming (data not shown). In Signaling in PMNs contrast, release of MMP-9 and β-glucuronidase was If CG and NE were required to properly regulate β2 inte- normal after plating on fibrinogen (Figure S5). Cross- grin functions, one would predict that intracellular sig- β linking with anti-CD11b and anti-CD29 Abs (an anti- 1 naling events in CG/NE PMNs would also be affected. integrin Ab), but not anti-CD16 Abs or isotype control, In accordance with this notion, IC-activated CG/NE was sufficient to induce release of MIP-2 in wt PMNs PMNs exhibited increased total tyrosine phosphoryla- (Figure 3D). In contrast, CD11b and CD29 crosslinking tion (Figure 4D). The Syk tyrosine kinase is an essential in CG/NE PMNs failed to elicit a similar response (Fig- component of integrin signaling in neutrophils (Mocsai ure 3D). These and the above results suggest that CG et al., 2002), and signaling proteins downstream of inte- and NE have a critical role in regulating integrin-medi- grin-mediated Syk activation include the adaptor mole- ated responses in PMNs. cule Vav1. We found that phosphorylation of both Syk Cathepsin G Regulates Neutrophil Functions 683

Figure 3. Defective Adhesion-Dependent CG/NE PMN Functions Stimulated with Direct Integrin Ligands ROI production from purified PMNs plated on fibrinogen (A) or fibronectin (B) in the absence or presence of TNF-α. Note that dihydrorhodamine 123 measures total ROI production (intracellular and released). (C) PMNs were plated on anti-CD16, anti-CD11b, and anti-CD29 Abs (40 ␮g/ml) or isotype control. Supernatant was collected after 2 hr and assayed for MIP-2. Data are representative of at least three independent experiments. All values represent mean ± SD. and Vav1 was deregulated in the absence of CG and for optimal MIP-2 release and ROI production and that NE (Figures 4D and 4E). In addition, IC-induced activa- possibly integrin clustering and cytoskeletal reorgani- tion of β2 integrins requires phosphoinositide 3-kinase zation are critical for these functions. To specifically (PI3-K) and is known to induce phosphorylation of pax- evaluate the role of adhesion in PMN effector functions, illin (Chen et al., 2003). Phosphorylation of these mole- we turned to a system in which we could stimulate cules was likewise increased in CG/NE PMNs (Figure PMNs to adhere without undergoing cytoskeletal re- 4D). In contrast, activation of the GTPase Rac1 was sig- arrangements. Mn2+ treatment of PMNs increases inte- nificantly decreased in CG/NE PMNs (Figure 4F). Inte- grin affinity directly, leading to cellular adhesion; how- grin clustering leads to activation of the Rho family of ever, in the absence of Ca2+, there is no cytoskeletal GTPases, which are known to be key regulators of the reorganization (Leitinger et al., 2000). Indeed, Mn2+- actin cytoskeleton (Burridge and Wennerberg, 2004). treated wt PMNs adhered efficiently to IC-coated sur- Defective Rac1 activation is therefore consistent with faces; however, there was little cell spreading and the absence of lamellipodia formation and cell spread- markedly diminished CD11b clustering (Figure 5A). ing observed in CG/NE PMNs. Thus, normal intracellu- Mn2+-treated wt PMNs also displayed a hyperphos- lar signaling downstream of integrins requires the pres- phorylation pattern when plated on IC or fibrinogen ence of CG and NE. (Figures 5B and 5C), suggesting a correlation between hyperphosphorylation and lack of cytoskeletal reorga- Cytoskeletal Reorganization Is Required for Optimal nization. In addition, Mn2+-treated, IC-stimulated wt PMN Effector Functions PMNs produced approximately 50% less ROI com- Our results suggest that in the in vitro IC-mediated sys- pared with wt PMNs stimulated in the presence of Ca2+ tem, FcγR ligation and PMN adhesion are insufficient and Mg2+ (Figure 5D). Lastly, Mn2+-treated, IC-stim- Immunity 684

Figure 4. Lack of Cell Spreading and Deregulated Intracellular Signaling in IC-Stimulated CG/NE PMNs (A) Labeled PMNs were plated on IC in the presence of TNF-α. At the indicated time points, the wells were washed, and the percentage of adherent cells remaining in the wells was measured. Data are representative of five independent experiments. (B) PMNs were plated for 1 hr on IC (in the presence of TNF-α), washed, and fixed with 2% paraformaldhehyde. CD11b staining was performed on nonpermeabilized cells. For F-actin visualization, the cells were permeabilized with 0.1% Triton-X 100. (C) Resting, TNF-α- or IC-stimulated PMNs were analyzed for surface levels of CD11b by flow cytometry. (D) PMNs were stimulated on IC for the indicated time, and cell lysates were probed for total tyrosine phosphorylation (PY), phosphorylated p85α, Paxillin, or Syk, and total Syk served as the loading control for protein content. (E) PMNs were stimulated on IC for the indicated time, and Vav1 was immunoprecipitated with anti-Vav1 Abs. Western blots were performed with Abs to PY or to Vav1. Vav1 phosphorylation was calculated from the ratio of anti-PY/total Vav1, and phosphorylation of wt PMNs at 15 min was set to 100%. Data are representative of two independent experiments. (F) Rac1-GTP was precipitated with PAK-1PBD after 15 and 30 min of IC stimulation or after in vitro GTPγS protein loading, which activates 100% of Rac1 in protein lysates and serves as an internal control for protein loading content. Rac1 activation was calculated from the ratio of IC-activated Rac1 and in vitro GTPγS loading, and Rac1 activation in wt at 15 min was set to 100%. Data are representative of three independent experiments. ulated wt PMNs did not release MIP-2 (Figure 5E). toskeletal rearrangements. Thus, cytoskeletal reorgani- These findings are not the result of a degranulation de- zation is critical for optimal MIP-2 and ROI production, fect, as release of NE from primary granules was normal independent of adhesion. Treatment of wt PMNs with in the presence of Mn2+ (Figure S6). Treatment of cells Mn2+ therefore recapitulates some of the activation de- with anti-integrin Abs to artificially induce clustering did fects seen in IC-stimulated CG/NE PMNs. These results not lead to MIP-2 production in the presence of Mn2+ are consistent with the apparent normal adhesion but (Figure 5F), suggesting that anti-integrin Abs can by- defective effector functions observed in CG/NE PMNs pass the requirement for high-affinity state but cannot and argue that CG’s and NE’s critical role is in the regu- lead to MIP-2 secretion in the absence of Ca2+ and cy- lation of cytoskeletal reorganization. Cathepsin G Regulates Neutrophil Functions 685

Figure 5. Cytoskeletal Reorganization Is Critical for ROI and MIP-2 Production (A) PMNs were stimulated on IC in the presence of Ca2+ and Mg2+ or Mn2+ and stained for CD11b or F-actin. PMNs were activated on IC (B) or fibrinogen (C) in the presence of Ca2+ and Mg2+ or Mn2+ for the indicated times, and cell lysates were probed for total PY. Syk served as the protein-loading control. ROI production (D) and MIP-2 release by wt PMNs plated on IC (E) or on anti-integrin Abs (F) in the presence of Ca2+ and Mg2+ or Mn2+. MIP-2 on anti-integrin Abs was assayed at 2 hr. In (D)–(F), all values represent mean ± SD.

Serine Proteases Are Not Required for Integrin used as test ligands. Activated CD11b binds to the High-Affinity Conformational Changes complement component iC3b (Berman and Muller, Normal adhesion of CG/NE PMNs to IC-coated sur- 1995; Jin and Li, 2002), and the extent of binding is faces suggests that CD11b has undergone a conforma- reflected in the number of rosette-forming PMNs. As tional change to the high-affinity state upon IC activation. shown in Figure 6, a small percentage of PMNs plated To further explore this notion, we used an experimental on OVA formed rosettes (9% ± 3% for wt and 11% ± system in which we could directly assess the CD11b 5% for CG/NE). The percentage of rosette-forming cells affinity state for ligand binding. In these experiments, was significantly increased when PMNs were plated on sheep erythrocytes were coated with iC3b (EiC3b) and IC in the presence of Mn2+ (94% ± 5% for wt and Immunity 686

Figure 6. IC-Stimulated PMNs Mediate Rosette Formation with EiC3b (A) PMNs were plated on OVA or IC in the presence of Ca2+ and Mg2+ or Mn2+, and rosetting was performed as described in Experimental Procedures. In some experiments, anti-CD11b antibody was added and allowed to incubate for 30 min prior to addition of EiC3b. (B) Percentage of adherent cells with rosettes. Closed arrowheads indicate PMNs with rosettes; open arrowheads indicate PMNs without rosettes, and the thin arrow indicates unbound erythrocyte. Note the flattened appearance of wt PMNs plated on IC compared with the more rounded appearance of CG/NE PMNs. At least 200 PMNs were counted, and percent average represents mean ± SD from four randomly chosen fields.

93% ± 1% for CG/NE). IC stimulation in the presence blocking anti-CD11b antibody prior to the addition of of Ca2+ and Mg2+ led to 86% ± 1% rosetting in CG/NE erythrocytes totally abrogated rosette formation (Figure PMNs, confirming that CD11b has been activated to 6A). These results suggest that CG and NE are dispens- the high-affinity state. Surprisingly, wt PMNs plated on able for high-affinity integrin conformational changes. IC in the presence of Ca2+ and Mg2+ have a lower number of rosettes (25% ± 9%), perhaps because CD11b in Exogenous Addition of Active Human Cathepsin G wt PMNs may have redistributed to points of contacts Reconstitutes PMN Effector Functions between cells and IC-coated surfaces and, therefore, The fact that serine proteases remain bound to surface was less available for binding EiC3b. Lastly, the rosetting membrane upon release from primary granules sug- is CD11b-specific, as preincubation of cells with a gests that they may modulate integrin clustering and Cathepsin G Regulates Neutrophil Functions 687

integrin-dependent signaling from the outside. If this with this interpretation, we showed that adhesion in- were true, then we should be able to restore MIP-2 reduced by Mn2+, an activator of high-affinity ligand bind- lease and ROI production by providing an exogenous ing, was insufficient to cause release of MIP-2 or to source of serine proteases. To test this hypothesis, we produce optimal levels of ROI in the absence of integrin added a physiologic dose of purified human CG (hCG) clustering and cell spreading. Although the release of to CG/NE PMNs stimulated with IC. Exogenously added prestored granule content minutes after IC stimulation hCG completely restored the release of MIP-2 to wt was normal in the CG/NE PMNs, sustained and optimal levels (Figure 7A). Similarly, the addition of exogenous release of newly synthesized MIP-2 and ROI over time hCG also restored ROI production to near normal levels may require cytoskeletal reorganization and the activa- in CG/NE PMNs (Figure 7B). To determine whether the tion of the Rho family GTPases (Bokoch and Diebold, catalytic activity of hCG is required for reconstitution, 2002; Burridge and Wennerberg, 2004). Indeed, data we performed the same experiments with inactivated suggest that the transport, maturation, and exocytosis hCG (ihCG). Inactivation of hCG by PMSF totally abol- of nascent granules as they emerge from the trans- ished the ability of the protease to reconstitute effector Golgi network depend on the cooperative action of functions in CG/NE PMNs (Figures 7A and 7B). To rule microtubules and actin filaments (Rudolf et al., 2001). out the possibility that ihCG could not reconstitute the In addition, optimal production of ROI requires the as- effector functions because of its inability to associate sembly of the NADPH oxidase complex composed of with the cell surface of PMNs, we performed immuno- cytosolic components recruited to membrane bound fluorescence on CG/NE PMNs incubated in the ab- components after Rac GTPase activation (Bokoch and sence or presence of hCG. Both active and inactive Diebold, 2002). Our data therefore place CG directly in forms of hCG were found to associate with the cell sur- a pathway that regulates Rac GTPase activation and face (Figure 7C), suggesting that the catalytic activity cytoskeletal reorganization (Figure 4F). of hCG is not required for membrane association but How does CG regulate integrin clustering and cy- is critical for the regulation of PMN effector functions. toskeletal rearrangements? We considered the possi- Strikingly, the addition of hCG alone, but not ihCG, also bility that CG modulates integrin activity directly through restored the clustering pattern of CD11b in CG/NE proteolytic modification of the extracellular domain of PMNs (Figures 7D and 7E), suggesting that the proteo- these receptors. In fact, previous studies have shown β lytic activity of CG is crucial for the reorganization of that NE and CG enhance the affinity of 3 integrin through the limited proteolysis of the α subunit (Si- surface β2 integrins in vitro. Tahar et al., 1997). Although our results suggest that CD11b in CG/NE PMNs was converted to an active Discussion conformation upon IC stimulation (Figure 6), limited proteolysis may further stabilize the high-affinity state, In this report, we identified a novel and important func- expose ligand-induced binding site (LIBS) epitopes, tion of neutrophil-derived serine protease CG as a criti- and promote clustering of integrins. To test this hypoth- cal regulator of PMN effector functions during IC stimu- esis, we turned to the human system, as there are no lation. We found that CG/NE PMNs have severe defects available antibodies that recognize high-affinity CD11b in MIP-2 release and ROI production. We showed that in the murine system. To this end, we treated purified CG/NE PMNs also exhibited significant defects in cell human PMNs to exogenous hCG and monitored for signaling, including increased phosphorylation of Syk new LIBS with a well-characterized monoclonal anti- and Vav1 as well as defective Rac1 activation. In addi- body (clone CBRM1/5) (Oxvig et al., 1999) that recog- tion, CG/NE PMNs failed to undergo cytoskeletal reor- nizes the active conformation of human CD11b. We ganization, although they adhered normally to IC- saw no evidence that hCG can directly expose new coated surfaces. Exogenously added, proteolytically LIBS when incubated with human PMNs in suspension active CG largely restored these defects. Together, (data not shown). In addition, we found no evidence these observations link neutrophil-derived serine prote- that CD11b on the surface of IC-activated murine ases to integrin-dependent, regulated chemokine se- PMNs was proteolytically processed by serine prote- cretion and ROI production. ases (data not shown). Thus, CG does not directly acti- Much attention has been paid to the “affinity modula- vate integrins. tion” models of integrin activation in which conforma- We also explored the notion that serine proteases tional changes of the extracellular domain of the het- may regulate integrin clustering indirectly through an- erodimers lead to rapid integrin-dependent adhesion other surface receptor. An intriguing class of receptors (Takagi and Springer, 2002). Although it is clear that af- on PMNs that may be specific targets of serine prote- finity modulation plays a central role in regulating adhe- ases are the protease-activated receptors (PARs). PARs sive properties of integrins, few studies have distin- are G protein-coupled receptors that undergo proteo- guished the precise role of “avidity modulation” or lytic cleavage of the N terminus, thus exposing a teth- clustering in integrin-mediated functions. However, ered ligand that subsequently autoactivates the recep- most of the issues over “affinity” versus “avidity” are tors (Coughlin and Camerer, 2003; O’Brien et al., 2001). considered in the context of cellular adhesion rather Among the four PARs described to date, PAR-2 is ex- than cellular effector functions. Although enhanced af- pressed on PMNs (Howells et al., 1997). To examine finity is sufficient for ligand binding and adhesion, we whether chemokine and ROI production by murine propose that subsequent clustering of integrins that PMNs is dependent on PAR-2 activation by CG, we leads to avidity modulation is essential for certain inte- sought to bypass the requirement for PAR-2 proteolytic grin-mediated neutrophil effector functions. Consistent cleavage by stimulating CG/NE PMNs directly with an Immunity 688

Figure 7. Exogenously Added hCG Restores PMN Functions Wt and CG/NE PMNs were plated on IC in the absence or presence of hCG or ihCG and assayed for MIP-2 release (A) or ROI production (B) over time. Data represent the mean ± SD of at least two independent experiments. ROI data are presented as percentage of wt PMNs at 125 min. (C) hCG and ihCG both associate with cell surface; hCG (red) and CD11b (green). (D) hCG, but not ihCG, restored CD11b clustering in CG/NE PMNs. (E) Percentage of cells with CD11b clustering (defined as a punctate pattern seen around at least 50% of the cell contact points). At least 400 cells per condition were individually counted and the values represent mean ± SD as determined by two blinded observers. (F) A model of CG-regulating PMN effector functions. IC stimulation and FcγR ligation leads to degranulation and increased integrin affinity. CG released from the cell remains bound to the surface membrane and regulates integrin clustering, Rac1 activation, and cytoskeletal reorganization that subsequently leads to ROI production and MIP-2 secretion. Mn2+ increases integrin affinity directly but does not lead to cytoskeletal reorganization in the absence of Ca2+. Anti-integrin Abs can bypass the high affinity requirement for integrin ligand binding but does not lead to cytoskeletal reorganization in the absence of CG. agonist peptide, SLIGRL (Moffatt et al., 2002). However, monocytes and PMNs through the fMLP receptor. How- we did not detect any enhancement of MIP-2 secretion ever, our findings that PTX, a G protein-coupled recep- or ROI production with the addition of this peptide ago- tor inhibitor, did not affect MIP-2 release in wt or CG/ nist (data not shown). Another CG receptor on PMNs NE PMNs while significantly inhibiting chemotaxis (Fig- that was recently identified is the G protein-coupled ure S3) suggests that these two functions require dif- fMLP receptor (Sun et al., 2004). In the human system, ferent signaling pathways. CG can act as an agonist to induce chemotaxis of Finally, these in vitro data of serine protease modula- Cathepsin G Regulates Neutrophil Functions 689

tion of PMN effector functions offer a possible explana- al., 2002). PMN purity was 75%–85% as assessed by forward and tion for the in vivo observations of impaired PMN re- side scatter with flow cytometry. cruitment and decreased chemokine production in the In Vitro PMN Stimulation air pouch. The initial PMNs recruited to the site of IC- ICs were formed by coating tissue culture plates with 5 mg/ml OVA induced inflammation release serine proteases that (Sigma A5503) overnight at 4°C followed by rabbit anti-OVA serum help regulate PMN effector functions in an autocrine (Sigma C6534) at 25 ␮g/ml (unless otherwise noted). Alternatively, fashion. The chemokines secreted by PMNs promote plates were coated with fibrinogen (200 ␮g/ml, Sigma F9754), fibro- further influx of leukocytes and activate proximal cells nectin (200 ␮g/ml, Sigma F0895), 40 ␮g/ml of anti-CD16 (Phar- to release more proinflammatory factors. In addition to mingen 553142), anti-CD11b (Serotec MCA711), anti-CD29 (Phar- mingen 553715) Abs, or rat IgG2a as isotype control (Pharmingen this autocrine regulation of PMN functions, serine pro- 553926) and blocked with 5 mg/ml OVA. Purified PMNs (at5×106 teases themselves have been shown in vitro to cause cell/ml) in X-VIVO 10 Media (BioWhittaker 04-380Q) or KRP buffer cytokine and chemokine release from resident cells (PBS, 1 mM Ca2+, 1.5 mM Mg2+, and 5.5 mM glucose) were plated such as macrophages and epithelial cells (Coeshott et on IC at 300 ␮l per well in 24-well plates in the presence of 10 ng/ al., 1999; Fadok et al., 2001; Sugawara et al., 2001). The ml TNF-α. In some experiments, PMNs were stimulated on IC in the 2+ 2+ 2+ direct or indirect effect of serine proteases on resident presence of 1 mM Mn instead of Ca and Mg and incubated at 37°C for the indicated times. As controls, PMNs were stimulated cells may explain the substantially higher levels of CXC with 200 ng/ml of PMA. chemokines released into the air pouch. In addition, ROI can also contribute to inflammation by causing Chemokine Analysis metabolic dysfunction and tissue damage (Mylonas Concentrations of MIP-2 were determined by ELISA (R&D Systems) and Kouretas, 1999); the overall effects are to generate and carried out as described in the manufacturer’s protocols. PMN more inflammatory signals. CG/NE PMNs lack the po- pellets were resuspended in 300 ␮l of PBS and 0.1% Tween 20, tential to sustain optimal levels of chemokine release freeze-thawed three times, and cleared by centrifugation prior to ELISAs. Total amount of MIP-2 was calculated from the formula and ROI production, resulting in a severely blunted in- (MIP-2 in supernatant + MIP-2 in cell pellets). flammatory response or in early termination of the inflammatory response. MIP-2 Analysis by Flow Cytometry In summary, our results suggest a model in which IC Intracellular chemokine analysis and surface staining were per- activation leads to the release of granule-associated formed as previously reported (Dorner et al., 2002) with the follow- serine proteases CG and NE. The proteases remain ing modifications. Intracellular staining was performed with anti- bound to the cell surface where CG acts to increase MIP-2 Abs (R&D Systems, AF-452), and anti-Gr-1 Abs (Pharmigen 553128) were used for PMN surface staining. integrin clustering and to enhance integrin-mediated functions in PMNs (Figure 7F). Identification of this CG Western Blot Analysis substrate would likely yield significant insights into ser- Activated PMNs were lysed directly in 2× SDS sample buffer, boiled ine protease functions and integrin activation. Nonethe- for 5 min, and fractionated with 10% sodium dodecyl-sulfate-poly- less, our findings of CG’s role in PMN activation raise acrylamide gel electrophoreses (SDS-PAGE). The blots were unexpected and interesting questions regarding the probed for total phosphotyrosine (4G10, Upstate 05-321), phospho-Syk (Tyr323/525/526, Cell Signaling 2711 and 2715), phospho- general role of serine proteases in integrin-mediated PI 3-kinase p85α (Tyr508, Santa Cruz SC-12929), phospho-Paxillin cellular functions in general. Whereas CG is essentially (Ser178, Bethyl Laboratories A300-100), or Syk (Santa Cruz N-19) PMN-specific, similar serine proteases are found in followed by the appropriate peroxidase-conjugated secondary another cell types (i.e., mast cell chymase). Because inte- tibodies (Jackson ImmunoResearch). Three million PMNs were grins are widely expressed cellular receptors, we envi- lysed in RIPA-based buffer (Mocsai et al., 2002), immunoprecipi- sion that this novel view of integrin avidity modulation tated with anti-Vav1 Abs (Santa Cruz H-211), and probed with 4G10 and anti-Vav1 Abs followed by the respective secondary Abs. by serine proteases may be relevant in other cell sys- PMNs were activated on IC, and Rac1 activation was performed tems as well. according to the manufacturer’s instructions (Upstate Biotechnol- ogy 17-283). Experimental Procedures Respiratory Burst Mice ROI was measured with OxyBURST Green H2HFFBSA (Molecular CG−/−, NE−/−, and CG/NE mice on the 129 genetic background were Probes O-13291) or Dihydrorhodamine 123 (Molecular Probes generated as previously described (Belaaouaj et al., 1998; MacIvor D-632) and carried out according to the manufacturer’s instruc- et al., 1999). These mice and wt control mice were kept in patho- tions. PMNs were resuspended at 5 × 106 cell/ml with 10 ng/ml gen-free conditions at Washington University Specialized Research TNF-α and plated in triplicates in 96-well plates at 100 ␮l per well. Facility. For in vitro analysis, mice 5–8 weeks of age were used. For Fluorescence was measured over time at 480 nm excitation and in vivo analysis, mice 6–8 weeks of age were used. 520 nm emission wavelengths with a FL500 microplate fluorescence reader (BIO-TEK Instruments). Specific ROI production was calculated with the following formula: [Fluorescence generated by Subcutaneous Air Pouch Model cells plated on specific ligands − nonspecific fluorescence gener- Air pouches were formed as previously described. All in vivo exper- ated by cells plated on OVA or anti-OVA]. iments were preformed on day 6 air pouches. Reverse passive Ar- thus reaction and zymosan injection were performed as previously Adhesion Assay described (Adkison et al., 2002). After 4 hr, the number of leuko- Purified PMNs (5 × 106 cell/ml) were labeled with calcein AM (Mo- cytes was enumerated, and chemokine levels were assayed by lecular Probes C3100) at 5 ␮g/ml for 30 min at 37°C. Labeled cells specific ELISA (R&D Systems). at 5 × 105 in 100 ␮l of KRP buffer were plated on IC in 96-well plates with TNF-α (10 ng/ml). At the indicated time, wells were PMN Purification washed twice with PBS to remove nonadherent cells. The fluores- PMNs were isolated from bone marrow by discontinuous Percoll cence was measured at 480 nm excitation and 520 nm emission. (Amersham 17-0891) gradient as previously described (Adkison et The percentage of adherent cells was determined by the following Immunity 690

formula: [fluorescence remaining after wash/total fluorescence be- Baggiolini, M., Dewald, B., and Moser, B. (1997). Human chemo- fore wash] × 100. kines: an update. Annu. Rev. Immunol. 15, 675–705. Belaaouaj, A., McCarthy, R., Baumann, M., Gao, Z., Ley, T.J., Abra- Exogenous Reconstitution with Human CG ham, S.N., and Shapiro, S.D. (1998). Mice lacking neutrophil elas- Human CG (hCG) (Elastin Products SG623) and inactivated hCG tase reveal impaired host defense against gram negative bacterial (ihCG) were added at the beginning of the assays at a final concen- sepsis. Nat. Med. 4, 615–618. tration of 1 ␮g/ml for MIP-2 reconstitution or 2 ␮g/ml for ROI recon- Berman, M.E., and Muller, W.A. (1995). Ligation of platelet/endothe- stitution. hCG was inactivated with 2 mM phenylmethanesulfonyl lial cell adhesion molecule 1 (PECAM-1/CD31) on monocytes and fluoride (PMSF) in 0.1 M Tris (pH 8) for 2 hr at 37oC and dialyzed neutrophils increases binding capacity of leukocyte CR3 (CD11b/ for 4 hr to overnight in 0.1 M Tris (pH 8). The remaining enzymatic CD18). J. Immunol. 154, 299–307. activity was determined by using the CG substrate N-succinyl-Ala- Ala-Pro-Phe-pNA as previously described (Adkison et al., 2002). Bokoch, G.M., and Diebold, B.A. (2002). Current molecular models for NADPH oxidase regulation by Rac GTPase. Blood 100, 2692– 2696. Fluorescent Microscopy LAB-TEK permanox chamber slides (Nalge Nunc) were coated with Burridge, K., and Wennerberg, K. (2004). Rho and Rac take center IC, and PMNs were plated on slides in the presence of TNF-α. After stage. Cell 116, 167–179. 1 hr, slides were washed twice with PBS and fixed with 2% parafor- Carden, D.L., and Korthuis, R.J. (1996). Protease inhibition attenu- maldehyde for 20 min. PMNs were stained for F-actin with Texas ates microvascular dysfunction in postischemic skeletal muscle. Red-X phalloidin (Molecular Probes T-7471). Surface staining for Am. J. Physiol. 271, H1947–H1952. CD11b (Pharmingen 557396) was performed on nonpermeabilized, Carney, D.F., Jagels, M.A., Hugli, T.E., Sands, H., and Rubin, H. paraformaldehyde-fixed cells. hCG staining was performed with (1998). Effect of serine proteinase inhibitors on neutrophil function: guinea pig anti-human CG (a generous gift from Tim Ley) followed alpha-1-proteinase inhibitor, antichymotrypsin, and a recombinant by rhodamine red-X-conjugated donkey anti-guinea pig (Jackson hybrid mutant of antichymotrypsin (LEX032) modulate neutrophil ImmunoResearch 706-295-148). All images were visualized on a Ni- adhesion interactions. J. Leukoc. Biol. 63, 75–82. kon Eclipse microscope and acquired with Magnafire software Chen, H., Mocsai, A., Zhang, H., Ding, R.X., Morisaki, J.H., White, (Optronics). M., Rothfork, J.M., Heiser, P., Colucci-Guyon, E., Lowell, C.A., et al. Rosetting Assay iC3b-coated sheep erythrocytes (EiC3b) were (2003). Role for plastin in host defense distinguishes integrin sig- prepared as previously described (Jin and Li, 2002). Briefly, anti- naling from cell adhesion and spreading. Immunity 19, 95–104. body-sensitized sheep erythrocytes (Advanced Research Technol- ogies B200) were washed three times in DGVB2+ (2.5 mM veronal Coeshott, C., Ohnemus, C., Pilyavskaya, A., Ross, S., Wieczorek, buffer [pH 7.5], 75 mM NaCl, 2.5% dextrose, 0.05% gelatin, 0.5 mM M., Kroona, H., Leimer, A.H., and Cheronis, J. (1999). Converting enzyme-independent release of tumor necrosis factor alpha and IL- MgCl2, 0.15 mM CaCl2), incubated with 20% mouse serum for 1 hr at 37°C, and washed three times with DGVB2+.5×107 EiC3b were 1beta from a stimulated human monocytic cell line in the presence added to IC-stimulated PMNs in the presence of Ca2+ and Mg2+ or of activated neutrophils or purified proteinase 3. Proc. Natl. Acad. Mn2+-containing buffers for 30 min at 37°C in a total volume of 250 Sci. USA 96, 6261–6266. ␮l. In some experiments, anti-CD11b Abs (1 ␮g/ml) were added to Coughlin, S.R., and Camerer, E. (2003). PARticipation in inflamma- the PMNs prior to EiC3b addition and allowed to bind for 30 min at tion. J. Clin. Invest. 111, 25–27. iC3b room temperature. After unbound E were washed away, the DeMali, K.A., Wennerberg, K., and Burridge, K. (2003). Integrin sig- cells were fixed in 2.5% gluteraldehyde, and the number of rosettes naling to the actin cytoskeleton. Curr. Opin. Cell Biol. 15, 572–582. (determined as PMNs with at least three attached EiC3b’s) was de- Dorner, B.G., Scheffold, A., Rolph, M.S., Huser, M.B., Kaufmann, termined by counting at least 200 PMNs in four randomly chosen S.H., Radbruch, A., Flesch, I.E., and Kroczek, R.A. (2002). MIP-1al- fields. pha, MIP-1beta, RANTES, and ATAC/lymphotactin function together with IFN-gamma as type 1 cytokines. Proc. Natl. Acad. Sci. USA 99, 6181–6186. Supplemental Data Fadok, V.A., Bratton, D.L., Guthrie, L., and Henson, P.M. (2001). Dif- Supplemental Data including six figures are available online with ferential effects of apoptotic versus lysed cells on macrophage this article at http://www.immunity.com/cgi/content/full/22/6/679/ production of cytokines: role of proteases. J. Immunol. 166, 6847– DC1/. 6854. Fearon, D.T., and Austen, K.F. (1977). Activation of the alternative Acknowledgments complement pathway due to resistance of zymosan-bound. Proc. Natl. Acad. Sci. USA 74, 1683–1687. The authors thank Ying Hu for excellent technical assistance, and Hirche, T.O., Atkinson, J.J., Bahr, S., and Belaaouaj, A. (2004). Defi- Hector Molina (Washington University) and Sherman Fong (Genen- ciency in neutrophil elastase does not impair neutrophil recruitment tech, Inc.) for helpful discussions and critically reading the manu- to inflamed sites. Am. J. Respir. Cell Mol. Biol. 30, 576–584. script. We thank Kathy Liszewski and Wojciech Swat for expert Hogg, N., Henderson, R., Leitinger, B., McDowall, A., Porter, J., and technical advice and reagents. We thank Tim Ley and Wayne Yo- Stanley, P. (2002). Mechanisms contributing to the activity of inte- koyama for their continued support. This work was partially sup- grins on leukocytes. Immunol. Rev. 186, 164–171. ported by a Biomedical Science Award from the Arthritis Founda- Howells, G.L., Macey, M.G., Chinni, C., Hou, L., Fox, M.T., Harriott, tion (C.T.N.P) and National Institutes of Health grants AI49261, P., and Stone, S.R. (1997). Proteinase-activated receptor-2: expres- P30AR48335 (C.T.N.P.), and HL54853 (S.D.S). sion by human neutrophils. J. Cell Sci. 110, 881–887. Huber, A.R., and Weiss, S.J. (1989). Disruption of the subendothe- Received: August 10, 2004 lial basement membrane during neutrophil diapedesis in an in vitro Revised: March 19, 2005 construct of a blood vessel wall. J. Clin. Invest. 83, 1122–1136. Accepted: March 23, 2005 Published: June 21, 2005 Jin, T., and Li, J. (2002). Dynamitin controls Beta 2 integrin avidity by modulating cytoskeletal constraint on integrin molecules. J. Biol. Chem. 277, 32963–32969. References Jones, S.L., Knaus, U.G., Bokoch, G.M., and Brown, E.J. (1998). Adkison, A.M., Raptis, S.Z., Kelley, D.G., and Pham, C.T. (2002). Two signaling mechanisms for activation of alphaM beta2 avidity in Dipeptidyl peptidase I activates neutrophil-derived serine prote- polymorphonuclear neutrophils. J. Biol. Chem. 273, 10556–10566. ases and regulates the development of acute experimental arthritis. Kessel, J.M., Hayflick, J., Weyrich, A.S., Hoffman, P.A., Gallatin, M., J. Clin. Invest. 109, 363–371. McIntyre, T.M., Prescott, S.M., and Zimmerman, G.A. (1998). Coen- Cathepsin G Regulates Neutrophil Functions 691

gagement of ICAM-3 and Fc receptors induces chemokine secre- ing transport, cortical restriction, and F-actin-dependent tethering. tion and spreading by myeloid leukocytes. J. Immunol. 160, 5579– Mol. Biol. Cell 12, 1353–1365. 5587. Scapini, P., Lapinet-Vera, J.A., Gasperini, S., Calzetti, F., Bazzoni, Laudanna, C., Kim, J.Y., Constantin, G., and Butcher, E. (2002). F., and Cassatella, M.A. (2000). The neutrophil as a cellular source Rapid leukocyte integrin activation by chemokines. Immunol. Rev. of chemokines. Immunol. Rev. 177, 195–203. 186, 37–46. Shappell, S.B., Toman, C., Anderson, D.C., Taylor, A.A., Entman, Leitinger, B., McDowall, A., Stanley, P., and Hogg, N. (2000). The M.L., and Smith, C.W. (1990). Mac-1 (CD11b/CD18) mediates ad- regulation of integrin function by Ca(2+). Biochim. Biophys. Acta herence-dependent hydrogen peroxide production by human and 1498, 91–98. canine neutrophils. J. Immunol. 144, 2702–2711. Li, Q., Park, P.W., Wilson, C.L., and Parks, W.C. (2002). Matrilysin Si-Tahar, M., Pidard, D., Balloy, V., Moniatte, M., Kieffer, N., Van shedding of syndecan-1 regulates chemokine mobilization and Dorsselaer, A., and Chignard, M. (1997). Human neutrophil elastase transepithelial efflux of neutrophils in acute lung injury. Cell 111, proteolytically activates the platelet integrin alphaIIbbeta3 through 635–646. cleavage of the carboxyl terminus of the alphaIIb subunit heavy chain. Involvement in the potentiation of platelet aggregation. J. Liu, Z., Zhou, X., Shapiro, S.D., Shipley, J.M., Twining, S.S., Diaz, Biol. Chem. 272, 11636–11647. L.A., Senior, R.M., and Werb, Z. (2000). The serpin alpha1-proteinase inhibitor is a critical substrate for gelatinase B/MMP-9 in vivo. Sugawara, S., Uehara, A., Nochi, T., Yamaguchi, T., Ueda, H., Sugiy- Cell 102, 647–655. ama, A., Hanzawa, K., Kumagai, K., Okamura, H., and Takada, H. (2001). Neutrophil proteinase 3-mediated induction of bioactive IL- MacIvor, D.M., Shapiro, S.D., Pham, C.T., Belaaouaj, A., Abraham, 18 secretion by human oral epithelial cells. J. Immunol. 167, 6568– S.N., and Ley, T.J. (1999). Normal neutrophil function in cathepsin 6575. G-deficient mice. Blood 94, 4282–4293. Sun, R., Iribarren, P., Zhang, N., Zhou, Y., Gong, W., Cho, E.H., Mackarel, A.J., Cottell, D.C., Russell, K.J., FitzGerald, M.X., and Lockett, S., Chertov, O., Bednar, F., Rogers, T.J., et al. (2004). Identi- O’Connor, C.M. (1999). Migration of neutrophils across human pul- fication of neutrophil granule protein cathepsin G as a novel che- monary endothelial cells is not blocked by matrix metalloproteinase motactic agonist for the G protein-coupled formyl peptide receptor. or serine protease inhibitors. Am. J. Respir. Cell Mol. Biol. 20, J. Immunol. 173, 428–436. 1209–1219. Takagi, J., and Springer, T.A. (2002). Integrin activation and struc- Mocsai, A., Zhou, M., Meng, F., Tybulewicz, V.L., and Lowell, C.A. tural rearrangement. Immunol. Rev. 186, 141–163. (2002). Syk is required for integrin signaling in neutrophils. Immu- Tang, T., Rosenkranz, A., Assmann, K.J., Goodman, M.J., Gutierrez- nity 16, 547–558. Ramos, J.C., Carroll, M.C., Cotran, R.S., and Mayadas, T.N. (1997). Moffatt, J.D., Jeffrey, K.L., and Cocks, T.M. (2002). Protease-acti- A role for Mac-1 (CDIIb/CD18) in immune complex-stimulated neu- vated receptor-2 activating peptide SLIGRL inhibits bacterial lipo- trophil function in vivo: Mac-1 deficiency abrogates sustained polysaccharide-induced recruitment of polymorphonuclear leuko- Fcgamma receptor-dependent neutrophil adhesion and comple- cytes into the airways of mice. Am. J. Respir. Cell Mol. Biol. 26, ment-dependent proteinuria in acute glomerulonephritis. J. Exp. 680–684. Med. 186, 1853–1863. Mylonas, C., and Kouretas, D. (1999). Lipid peroxidation and tissue Tkalcevic, J., Novelli, M., Phylactides, M., Iredale, J.P., Segal, A.W., damage. In Vivo 13, 295–309. and Roes, J. (2000). Impaired immunity and enhanced resistance O’Brien, P.J., Molino, M., Kahn, M., and Brass, L.F. (2001). Protease to endotoxin in the absence of neutrophil elastase and cathepsin activated receptors: theme and variations. Oncogene 20, 1570– G. Immunity 12, 201–210. 1581. Woodman, R.C., Reinhardt, P.H., Kanwar, S., Johnston, F.L., and Olson, T.S., and Ley, K. (2002). Chemokines and chemokine recep- Kubes, P. (1993). Effects of human neutrophil elastase (HNE) on tors in leukocyte trafficking. Am. J. Physiol. Regul. Integr. Comp. neutrophil function in vitro and in inflamed microvessels. Blood 82, Physiol. 283, R7–28. 2188–2195. Owen, C.A., and Campbell, E.J. (1999). The cell biology of leuko- Yamada, K.M., and Miyamoto, S. (1995). Integrin transmembrane cyte-mediated proteolysis. J. Leukoc. Biol. 65, 137–150. signaling and cytoskeletal control. Curr. Opin. Cell Biol. 7, 681–689. Zhou, M.J., and Brown, E.J. (1994). CR3 (Mac-1, alpha M beta 2, Owen, C.A., Campbell, M.A., Boukedes, S.S., and Campbell, E.J. CD11b/CD18) and Fc gamma RIII cooperate in generation of a neu- (1995a). Inducible binding of bioactive cathepsin G to the cell sur- trophil respiratory burst: requirement for Fc gamma RIII and tyro- face of neutrophils. A novel mechanism for mediating extracellular sine phosphorylation. J. Cell Biol. 125, 1407–1414. catalytic activity of cathepsin G. J. Immunol. 155, 5803–5810. Owen, C.A., Campbell, M.A., Sannes, P.L., Boukedes, S.S., and Campbell, E.J. (1995b). Cell surface-bound elastase and cathepsin G on human neutrophils: a novel, non-oxidative mechanism by which neutrophils focus and preserve catalytic activity of serine proteinases. J. Cell Biol. 131, 775–789. Oxvig, C., Lu, C., and Springer, T.A. (1999). Conformational changes in tertiary structure near the ligand binding site of an integrin I domain. Proc. Natl. Acad. Sci. USA 96, 2215–2220. Ravetch, J.V. (1994). Fc receptors: rubor redux. Cell 78, 553–560. Reeves, E.P., Lu, H., Jacobs, H.L., Messina, C.G., Bolsover, S., Ga- bella, G., Potma, E.O., Warley, A., Roes, J., and Segal, A.W. (2002). Killing activity of neutrophils is mediated through activation of proteases by K+ flux. Nature 416, 291–297. Rezzonico, R., Imbert, V., Chicheportiche, R., and Dayer, J.M. (2001). Ligation of CD11b and CD11c beta(2) integrins by antibodies or soluble CD23 induces macrophage inflammatory protein 1al- pha (MIP-1alpha) and MIP-1beta production in primary human monocytes through a pathway dependent on nuclear factor-kap- paB. Blood 97, 2932–2940. Rudolf, R., Salm, T., Rustom, A., and Gerdes, H.H. (2001). Dynamics of immature secretory granules: role of cytoskeletal elements dur-