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Molecular Immunology Molecular Immunology Document Transcript

  • Molecular Immunology 47 (2010) 894–902 Contents lists available at ScienceDirect Molecular Immunology journal homepage: Short communication Calpain inhibition impairs TNF- -mediated neutrophil adhesion, arrest and oxidative burst Andrew J. Wiemer a,b,1 , Mary A. Lokuta c,2 , Jill C. Surfus a,1 , Sarah A. Wernimont a,1 , Anna Huttenlocher a,d,∗ a Department of Medical Microbiology and Immunology, University of Wisconsin, Madison, WI, USA b Institute on Aging, University of Wisconsin, Madison, WI, USA c Stratatech Corporation, Suite 169, 505 S. Rosa Road, Madison, WI 53719, USA d Pediatrics, University of Wisconsin, Madison, WI, USA a r t i c l e i n f o a b s t r a c t Article history: Proinflammatory cytokines, such as tumor necrosis factor alpha (TNF- ), are increased in many chronic Received 3 August 2009 inflammatory disorders, including rheumatoid arthritis, and contribute to recruitment of neutrophils Received in revised form 5 October 2009 into areas of inflammation. TNF- induces a stop signal that promotes neutrophil firm adhesion and Accepted 6 October 2009 inhibits neutrophil polarization and chemotaxis. Calpain is a calcium-dependent protease that mediates Available online 3 November 2009 cytoskeletal reorganization during cell migration. Here, we show that calpain inhibition impairs TNF- - induced neutrophil firm adhesion to fibrinogen-coated surfaces and the formation of vinculin-containing Keywords: focal complexes. Calpain inhibition induces random migration in TNF- -stimulated cells and prevents Migration Stop signal the generation of reactive oxygen species, but does not alter TNF- -mediated activation of p38 MAPK Neutrophil and ERK MAPK. These findings suggest that the TNF- -induced neutrophil arrest requires the activity of Oxidative burst calpain independent of p38 MAPK and ERK signaling seen after TNF- stimulation. Together, our data Tumor necrosis factor suggest that therapeutic inhibition of calpain may be beneficial for limiting TNF- -induced inflammatory Motility responses. © 2010 Elsevier Ltd. All rights reserved. 1. Introduction studying neutrophil migration is important for understanding the mechanisms that mediate the development of acute and The inflammatory response is essential for proper wound heal- chronic inflammation. Development of agents that either selec- ing (Gillitzer and Goebeler, 2001); however, in conditions including tively inhibit or activate neutrophil migration represents an asthma, rheumatoid arthritis and inflammatory bowel disease intriguing approach for the treatment of inflammatory disorders excessive inflammation contributes to the development of chronic (Mackay, 2008). inflammation and tissue damage (Aloisi and Pujol-Borrell, 2006). Tumor necrosis factor alpha (TNF- ) is a central mediator of Neutrophils are early responders to inflammatory stimuli including the inflammatory response that is released by macrophages and cytokines, chemokines and extracellular matrix ligands (Mayadas other cells in response to a variety of proinflammatory stimuli and Cullere, 2005). The inflammatory response involves neu- (Locksley et al., 2001). Elevated levels of TNF- are often associ- trophil adhesion to and migration out of the microvasculature ated with chronic inflammation, and there are therapeutic benefits into the affected tissue, and the subsequent retention of neu- to blocking TNF- with agents such as etanercept or infliximab trophils within tissues (Granger and Kubes, 1994). Consequently, in patients with chronic inflammatory disorders (Aggarwal, 2003). TNF- causes a cascade of events including cytokine and chemokine production, proliferation of immune cells, and increased cell adhe- sion, thus recruiting and retaining leukocytes in inflammatory sites. Abbreviations: TNF- , tumor necrosis factor alpha; MAPK, mitogen activated TNF- activates several neutrophil functions including oxidative protein kinase; ICAM-1, intercellular adhesion molecule 1; FBG, fibrinogen. ∗ Corresponding author at: Department of Medical Microbiology and Immunol- burst (Nathan, 1987) and degranulation (van der Poll et al., 1992). ogy, University of Wisconsin, 4205 Microbial Sciences Building, 1550 Linden Avenue, While TNF- does not increase cytosolic free calcium levels (Richter Madison, WI 53706, USA. Tel.: +1 608 265 4642; fax: +1 608 262 8418. et al., 1989), the ability of TNF- to induce activation of neu- E-mail address: (A. Huttenlocher). trophils is dependent upon calcium release and can be blocked 1 Department of Medical Microbiology and Immunology, University of Wisconsin, by calcium chelators (Richter et al., 1990) or calcium channel 4225 Microbial Sciences Building, 1550 Linden Avenue, Madison, WI 53706, USA. Tel.: +1 608 265 4669; fax: +1 608 262 8418. blockers. TNF- also up-regulates the adhesion molecules CD11b 2 Tel.: +1 608 441 8358; fax: +1 608 441 2757. and CD15 on the surface of neutrophils and induces neutrophil 0161-5890/$ – see front matter © 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.molimm.2009.10.002
  • A.J. Wiemer et al. / Molecular Immunology 47 (2010) 894–902 895 arrest (Lokuta and Huttenlocher, 2005; Mayadas and Cullere, 2.2. Neutrophil isolation 2005). There are several well-characterized signaling pathways acti- Human peripheral blood neutrophils were purified from blood vated by TNF- binding to its two predominant receptors, TNFR1 using Polymorphprep from Nycomed (Zurich, Switzerland) accord- and TNFR2. Binding of TNF- leads to increased NF-kB activity ing to manufacturer protocol and as described previously (Lokuta and expression of its target genes, including increased expression et al., 2007). All donors were healthy and informed consent was of adhesion molecules E-selectin, VCAM, and ICAM-1 (Read et al., obtained at the time of the blood draw. The human subject proto- 1995). In addition, TNF- stimulation activates signaling through col was approved by the University of Wisconsin Center for Health MAPKs (mitogen activated protein kinases) including p38 and ERK Sciences Human Subjects Committee. (McLeish et al., 1998), and signaling through the small GTPase Cdc42 (Puls et al., 1999). Ultimately, TNF- signaling leads to inte- 2.3. Immunofluorescent staining grin activation and firm adhesion of neutrophils to a variety of Neutrophils were plated on glass coverslips coated with surfaces (Williams and Solomkin, 1999), via mechanisms that are 10 g/mL fibrinogen and incubated for 30 min at 37 ◦ C and 7.5% currently not well-characterized. CO2 in the presence of TNF- and/or calpain inhibitor. Cells Calpains are intracellular, calcium-dependent proteases (Franco were fixed with 6.6% paraformaldehyde, 0.05% glutaraldehyde, and and Huttenlocher, 2005) that are constitutively active in resting 0.25 mg/mL saponin in PBS for 15 min. This reaction was quenched neutrophils (Lokuta et al., 2003). Calpains contribute to polariza- with 0.15 M glycine in PBS for 15 min. Samples were blocked tion and formation of lamellipodia during chemotaxis (Nuzzi et al., with PBS containing 10% heat-inactivated FBS and 0.25 mg/mL 2007). In most cell types, calpain inhibition decreases migration saponin at 4 ◦ C for 24–48 h. Anti-vinculin antibody (V-9131; Sigma) (Huttenlocher et al., 1997), but in human neutrophils, calpain inhi- was used at 1:400 in blocking buffer. Anti-actin antibody (AC-15; bition enhances random neutrophil migration (Lokuta et al., 2003; Sigma) was used at 1:500, DAPI (Sigma) was used at 1:10,000, Katsube et al., 2008). Through cleavage of their substrates, calpains and FITC-anti-tubulin antibody (Sigma) was used at 1:200. Sheep- regulate activity of the Rho family of small GTPases (Kulkarni et al., anti-mouse-FITC from Valeant (Costa Mesa, CA) was used at 1999), activation of integrins (Stewart et al., 1998), and organiza- 1:250 or goat-anti-mouse-rhodamine Red X was used at 1:250 tion of the cytoskeleton (Huttenlocher et al., 1997). Inhibition of (Invitrogen (Carlsbad, CA)). Coverslips were imaged using a 100× calpains has been reported to induce activation of p38 MAPK and oil-immersion lens on a Nikon Eclipse TE300 inverted fluores- ERK MAPK under some circumstances (Katsube et al., 2008). Calpain cent microscope. Images were acquired with a Hamamatsu cooled activity reportedly is elevated in several human diseases including CCD video camera and captured into Metamorph v7.1 from MDS muscular dystrophy (Huang and Wang, 2001). Inhibition of calpain (Toronto, Canada). activity in an animal model of collagen-induced arthritis blocked neutrophil recruitment and prevented inflammation (Cuzzocrea et 2.4. Adhesion assay al., 2000). The signaling pathways involved with neutrophil migration are Neutrophils were incubated in EGM-2MV containing 1.93 M of complex (Niggli, 2003), and the regulation of neutrophil arrest by the cell permeable fluorescent indicator, calcein-AM (Invitrogen) TNF- is not well-characterized. In other processes and cell types, for 15 min at 37 ◦ C and 7.5% CO2 . Cells were then washed with PBS TNF- -induced signaling requires calpain activity for its effects. For without Ca2+ /Mg2+ . Neutrophils were brought to 1 × 106 cells/mL example, inhibitors of calpain reverse TNF- -induced apoptosis in in DPBS without Ca2+ /Mg2+ for the standard curve or brought to the U937 monocytic cell line (Vanags et al., 1996) and the Jurkat T 1 × 106 cells/mL in EGM-2MV and treated accordingly. Neutrophils lymphocytic cell line (Diaz and Bourguignon, 2000). In neutrophils, were allowed to adhere for 30 min on 2.5 g/mL fibrinogen- TNF- signaling has been reported to cause apoptosis through sep- or ICAM-1-coated, black, medium-binding 96-well plates from arate caspase- or calpain-dependent pathways (Chen et al., 2006). Greiner (Monroe, NC). Unbound cells were removed by washing Here, we investigated how calpain inhibition alters TNF- - and firm shaking. Fluorescence excitation/emission at 485/535 nm mediated neutrophil adhesion and migration. In this report, we was determined using a TECAN SPECTRAFluor Plus fluorometer. demonstrate that calpain inhibition alters several consequences of Samples were run in quadruplicate, a standard curve included on TNF- signaling in neutrophils. Treatment with calpain inhibitors each plate, and linear regression was performed with Magellan decreases TNF- -induced neutrophil firm adhesion to fibrinogen, v2.50 software to determine the number of neutrophils adhered induces random migration in TNF- -stimulated cells, and pre- in each well. vents TNF- -induced generation of reactive oxygen species. Taken together, the data indicate that calpain inhibition can impair TNF- 2.5. Oxidative burst assays -mediated neutrophil activation. Extracellular oxidative burst was assayed using homovanillic acid. Briefly, 100 L of 400 M homovanillic acid with 4 U/mL 2. Materials and methods horseradish peroxidase in HBSS and 75 L of EGM-2MV containing treatment were equilibrated to 7.5% CO2 and 37 ◦ C in black 96-well 2.1. Reagents plates. Then, 1 × 105 neutrophils were added in 20 L and incu- bated for 30 min at 7.5% CO2 and 37 ◦ C. For the last 10 min of the Recombinant human TNF- was purchased from R&D Sys- incubation 5 L of media or fMLP to a final concentration of 100 nM tems (Minneapolis, MN). Fibrinogen and f-Met-Leu-Phe (fMLP) was added. The plate was then spun, 100 L of the supernatant were purchased from Sigma Chemical Co. (St. Louis, MO). Calpain transferred to a clean well, and fluorescence excitation/emission at inhibitors ALLN, ALLM, Z-LLY-FMK, PD150606, and calpastatin pep- 355/405 determined using a Perkin Elmer Victor3 V 1420 Multilabel tide were purchased from EMD Biosciences (San Diego, CA). The Counter. calpain inhibitors used in this study were cell permeable peptide aldehydes ALLN (50 g/mL, 130 M) and ALLM (50 g/mL, 125 M) 2.6. Time-lapse microscopy (concentrations which inhibit ∼80% of calpain activity in neu- trophils (Lokuta et al., 2003)), the fluoromethyl ketone Z-LLY-FMK Neutrophils were plated for 30 min at 37 ◦ C and 7.5% CO2 (45 M), and the small molecule PD150606 (1 M). onto non-tissue-culture-treated dishes in EGM-2MV from Cam-
  • 896 A.J. Wiemer et al. / Molecular Immunology 47 (2010) 894–902 Fig. 1. Calpain mediates TNF- -induced cell adhesion. (A) Human peripheral blood neutrophils plated on fibrinogen (FBG) or intercellular adhesion molecule 1 (ICAM-1) (B) Neutrophils were treated for 30 min with TNF- (250 ng/mL) in the presence or absence of calpain inhibitors ALLM (M) or ALLN (N) (50 g/mL), Z-LLY-FMK (L) (45 M), or PD150606 (P) (1 M). Results are representative images of at least three experiments. Neutrophils were assessed for adhesion to (C) FBG or (D) ICAM-1 coated wells (mean ± SEM, n = 3, p < 0.05 by one-way ANOVA, Tukey post hoc analysis indicates significant difference relative to control (*) or TNF (#)). brex (East Rutherford, NJ) containing TNF- and/or inhibitor as gradient gels, transferred to nitrocellulose using standard meth- noted. Dishes were then placed in The Box closed system from ods, and blotted with anti-p38 MAPK or anti-phospho-p38 Life Imaging Services (Basel, Switzerland), and maintained at 37 ◦ C MAPK antibodies (Biosource). Detection was performed using and imaged on an Olympus (Center Valley, PA) IX-70 inverted Alexa-Fluor® 680 goat-anti-mouse IgG (Molecular Probes) and microscope using a 20× phase objective. Images were collected IRDyeTM 800CW goat-anti-rabbit IgG (Rockland) antibodies. Quan- using a Coolsnap fx cooled CCD camera from Photometrics (Tus- tification was determined using an Odyssey Infrared-Imaging can, AZ) and captured into MetaView v6.2 (MDS) every 15 s for System. 15 min. 2.7. Western blot analysis 2.8. Statistical analysis Neutrophils were stimulated with 250 ng/mL TNF- for Statistical analyses were performed using Graph Pad (Prism). 30 min at 37 ◦ C and lysed in 50 mM HEPES pH 7.4, 1% Triton Statistical significance was calculated using one-way or two-way X-100, 1 mM EDTA, and 1 mM EGTA using a method modi- analysis of variance (ANOVA) where indicated to assess for sig- fied from Suzuki et al. (1999). Lysing buffer also contained nificant differences in treatment and/or treatment day. Post hoc freshly added phosphatase inhibitor cocktail (1:50 dilution, analysis was performed using Tukey’s HSD. Data were normalized P-5726; Sigma), protease inhibitor cocktail (1:50 dilution, P- relative to the mean and expressed as fold increase relative to con- 8340; Sigma), 2 mM phenylmethylsulfonylfluoride (PMSF), 100 M trol. All columns in bar graphs represent the mean of the indicated sodium orthovanadate, 2 g/mL aprotinin, 2 g/mL leupeptin A, number of replicates. Error bars on graphs represent standard error 900 M benzamidine, 1 mM phenantroline, and 1 g/mL pepstatin of the mean (SEM). An level of 0.05 was set as the level of signif- A. Proteins were resolved by SDS-PAGE on 6–20% acrylamide icance. View slide
  • A.J. Wiemer et al. / Molecular Immunology 47 (2010) 894–902 897 Fig. 2. Calpain inhibition polarizes TNF- -treated cells. Human peripheral blood neutrophils were treated for 30 min with TNF- (50 ng/mL) in the presence of vehicle control or calpain inhibitors ALLN or ALLM (50 g/mL), Z-LLY-FMK (45 M), or PD150606 (1 M). Neutrophil actin arrangement was determined by rhodamine phalloidin staining. Results are representative images of at least three experiments. 3. Results vehicle control treated neutrophils retained a rounded morphol- ogy and appeared only weakly adherent to the fibrinogen (Fig. 1A). 3.1. Calpain inhibition decreases TNF-˛-induced neutrophil Control neutrophils exhibited stronger adhesion to ICAM-1 and dis- adhesion played polarized morphology (Fig. 1B). Following TNF- treatment neutrophils developed a non-polarized and spread morphology on Stimulation with TNF- induces firm neutrophil adhesion fibrinogen which was associated with increased adhesion (Lokuta (Lokuta and Huttenlocher, 2005). To determine whether calpain and Huttenlocher, 2005). Treatment with calpain inhibitors alone activity is required for TNF- -mediated adhesion, we treated changed the cell morphology of the adherent subpopulation from human peripheral blood neutrophils with TNF- alone (250 ng/mL) rounded to polarized (Lokuta et al., 2003). When added to TNF- - or in combination with a panel of calpain inhibitors and examined treated cells, calpain inhibitors decreased overall adhesion relative neutrophil adhesion to fibrinogen-coated or intercellular adhesion to TNF- alone and induced polarization. molecule 1 (ICAM-1)-coated coverslips (Fig. 1). Cells were allowed Cell adhesion assays were performed to quantify the effect of to adhere to fibrinogen-coated coverslips for 30 min in the pres- calpain inhibition on TNF- -mediated neutrophil adhesion (Fig. 1C ence of TNF- and/or indicated inhibitors. Cell morphology was and D). Cells were fluorescently labeled with calcein-AM, allowed initially assessed via light microscopy (Fig. 1A and B). As expected, to adhere to fibrinogen- or ICAM-1-coated 96-well plates for 30 min View slide
  • 898 A.J. Wiemer et al. / Molecular Immunology 47 (2010) 894–902 Fig. 3. Calpain inhibition causes altered organization of focal complexes and microtubules. Human peripheral blood neutrophils were treated for 30 min with TNF- (50 ng/mL) in the presence or absence of calpain inhibitor ALLN (50 g/mL). (A) Formation of focal complexes was assessed by the visualization of vinculin. Samples were co-stained with rhodamine phalloidin and DAPI. Results are representative images of at least three experiments. (B) Organization of microtubules was assessed by staining for tubulin. Samples were co-stained with DAPI. Results are representative images of at least three experiments. in the presence or absence of calpain inhibitors, and adhesion was increased baseline adhesion, and were not statistically significant. quantified by fluorescence detection. As expected, TNF- increased Therefore, further studies were performed using fibrinogen-coated adhesion of neutrophils to fibrinogen relative to vehicle controls surfaces. almost 10-fold. Calpain inhibitors alone had no statistically sig- Unstimulated neutrophils loosely adhere to fibrinogen and do nificant effect on adhesion. However, ALLN, ALLM, and PD150606 not polarize. We (Lokuta and Huttenlocher, 2005) and others significantly reduced the TNF- -mediated increase in adhesion (Fuortes et al., 1999) have shown that TNF- induces non- to fibrinogen. Z-LLY-FMK also appeared to reduce adhesion to polarized neutrophil spreading. Conversely, calpain inhibition in fibrinogen, although the results were not statistically significant. the absence of TNF- stimulation elicits polarized neutrophil Untreated neutrophils exhibited much stronger adhesion to ICAM- spreading (Lokuta et al., 2003). We therefore asked whether calpain 1 than to fibrinogen and consequently the TNF-mediated increase inhibition alters TNF- -induced cytoskeletal polarization. Cells was not as pronounced on this substrate. In addition, although were treated with TNF- in the presence or absence of calpain calpain inhibition also appeared to impair neutrophil adhesion inhibitors (Fig. 2) and assessed for actin architecture using fluo- to ICAM-1, these effects were more subtle, likely a result of the rescence microscopy. Vehicle control cells were loosely attached
  • A.J. Wiemer et al. / Molecular Immunology 47 (2010) 894–902 899 with rounded morphology and peripherally localized actin. As expected, neutrophils treated with TNF- exhibited non-polarized cell spreading and peripherally localized actin, while cells treated with calpain inhibitors alone exhibited polarized cell spreading with actin polarized to the leading edge. Calpain inhibition caused polarized neutrophil morphologies with actin reorganization to the leading edge even in the presence of TNF- . 3.2. Calpain inhibition alters cytoskeletal organization of adherent neutrophils Previous work has shown that treatment with TNF- induces the formation of non-polarized vinculin-containing focal com- plexes and increases distribution of NADPH oxidase to focal complexes (Yan et al., 1995). In order to determine whether calpain inhibition disrupts focal complex formation, cells were treated with TNF- in the presence or absence of calpain inhibitors and the formation of vinculin-containing focal complexes was examined (Fig. 3A). As expected, treatment with TNF- alone elicited the formation of non-polarized vinculin-containing focal complexes. In the presence of ALLN alone, the cells took on a polarized morphology with few vinculin-containing focal com- plexes. When neutrophils were treated with both TNF- and ALLN, cells exhibited an elongated and polarized morphology with few vinculin-containing focal complexes oriented to the cell rear. Similar results were observed with other calpain inhibitors including ALLM (data not shown). These data suggest that cal- pain inhibition impairs focal complex formation induced by TNF- . In order to further characterize cell polarization we exam- ined the effects of TNF- and calpain inhibitors on microtubule Fig. 4. Calpain inhibition reverses TNF- -mediated migration stop signal. Human organization. Neutrophils were treated with TNF- alone or in peripheral blood neutrophils were treated for 30 min with TNF- (50 ng/mL) in the combination with calpain inhibitor and tubulin orientation was presence or absence of calpain inhibitor ALLM (50 g/mL). Cell movement was cap- tured between 30 and 45 min post-treatment by time-lapse microscopy and cell assessed by fluorescence microscopy (Fig. 3B). In TNF- treated speeds were quantified using Metamorph (mean ± SEM). (A) ALLM increases dis- cells, formation of the microtubule organizing center (MTOC) is placement of TNF- treated neutrophils (data shown is representative of three evident, and the microtubules are not polarized but extend in separate experiments). (B) Calpain inhibitors ALLM, ALLN, PD150505 and LLYFMK all directions. However, in neutrophils treated with the calpain increase mean cell speeds of neutrophils treated with TNF- (data from three sep- arate experiments, *p < 0.01). inhibitor ALLN and TNF- , the MTOC localizes toward the rear of the cell with a distribution of microtubules which is typical of polarized 3.4. Calpain inhibition functions independently of TNF-induced neutrophils. MAPK activation 3.3. Calpain inhibition induces migration of TNF-˛-arrested Activation of p38 and ERK signaling occurs in neutrophils fol- neutrophils lowing TNF- stimulation (Suzuki et al., 1999), and p38 activation is required for TNF- -mediated neutrophil adhesion and arrest Several studies have indicated a role for TNF- signaling in (Lokuta and Huttenlocher, 2005). Additionally, calpain inhibition neutrophil migration (Spertini et al., 1991; Drost and MacNee, has been reported by Katsube et al. (2008) to induce p38 and ERK 2002). Our previous studies have demonstrated that TNF- stim- phosphorylation in human neutrophils. We investigated whether ulation induces neutrophil arrest and impairs neutrophil random calpain inhibition activates phosphorylation of p38 and ERK in migration (Lokuta and Huttenlocher, 2005). To determine if calpain combination with TNF- stimulation during the timeframe of our inhibition affects TNF- -mediated neutrophil arrest, neutrophils migration assays (30 min post-TNF- addition). Neutrophils were were treated for 30 min with 50 ng/mL TNF- in the presence treated with TNF- in the presence or absence of calpain inhibitors or absence of calpain inhibitors and migration was assessed and phosphorylation of p38 and ERK was assessed by Western Blot using time-lapse microscopy (Fig. 4). As expected, in the absence analysis (Fig. 5). TNF- induced an increase in levels of phospho- of TNF- , neutrophils were only loosely adherent and speeds rylated p38 and ERK at 15 and 30 min post-stimulation. Calpain of migration were not determined (for a full description of inhibition alone did not induce an increase in p38 and ERK phos- how calpain inhibition affects migration of unstimulated neu- phorylation relative to vehicle control under the conditions of this trophils see reference (Lokuta et al., 2003)). In the presence of assay. Inhibition of calpain activity also did not significantly alter TNF- , neutrophils migrated at a speed of 5.75 ± 0.50 m/min. TNF- -mediated p38 or ERK activation at these time points. These When neutrophils were treated with both TNF- and a calpain findings suggest that calpain inhibition likely affects neutrophil inhibitor, the speed of migrating cells increased. The effects were adhesion and migration in TNF- -treated neutrophils independent most pronounced for ALLN and ALLM and were over 2-fold, of p38 and ERK MAPK signaling. to 17.0 ± 1.16 and 15.0 ± 1.06 m/min, respectively. Both Z-LLY- FMK and PD150606 also significantly increased migration speeds 3.5. Calpain inhibitors prevent TNF-˛ mediated oxidative burst although to a lesser extent than ALLN and ALLM. Together, the data indicate that calpain inhibition blocks TNF- -mediated neutrophil One measure of neutrophil activation is the release of reactive arrest. oxygen species, which is promoted by TNF- stimulation, enhanced
  • 900 A.J. Wiemer et al. / Molecular Immunology 47 (2010) 894–902 2-fold relative to untreated control cells. Treatment with the cal- pain inhibitors alone had no statistically significant effect on the oxidative burst. Treatment with all four calpain inhibitors signif- icantly reduced TNF- -mediated hydrogen peroxide generation. These findings indicate that calpain activity is required for TNF- -mediated oxidative burst. 4. Discussion TNF- -targeted therapies are highly effective for the treatment of rheumatoid arthritis and other chronic inflammatory disorders. Here we provide evidence that calpain inhibition impairs TNF- -mediated adhesion and arrest of primary human neutrophils. We also show that calpain inhibition induces cell polarization and random migration in TNF- -stimulated cells and prevents the generation of oxidative burst, but does not alter TNF- -mediated phosphorylation of p38 and ERK. Together, our findings suggest that therapeutic inhibition of calpain may be beneficial for limiting TNF- -induced inflammatory responses and tissue retention. It is well-documented that TNF- induces integrin-mediated firm neutrophil adhesion to a variety of surfaces including fib- rinogen and ICAM-1 (Nathan, 1987; Read et al., 1995; Lokuta and Huttenlocher, 2005). While neutrophil binding to ICAM-1 is likely mediated through MAC-1 ( M 2) (Diamond and Springer, 1993), adhesion to fibrinogen may be mediated by both MAC-1 ( M 2) and the vitronectin receptor ( v 3) (Hendey et al., 1996). We demonstrate that treatment with a panel of calpain inhibitors decreases TNF- -mediated neutrophil adhesion. This effect is most pronounced on fibrinogen, likely because on ICAM-1 neutrophils Fig. 5. Calpain inhibition does not affect TNF-mediated phosphorylation of p38 and ERK. Neutrophils were treated for 30 min with TNF- (250 ng/mL) in the presence exhibited higher baseline adhesion in unstimulated controls. The or absence of calpain inhibitor ALLM (50 g/mL). Levels of phosphorylated and total use of a panel of calpain inhibitors is important since each inhibitor p38 were assessed by Western Blot analysis. (A) Representative Western blots and displays different specificities toward both calpains and other pro- quantification of cells treated for 0, 15, or 30 min (n = 3, *p < 0.05 relative to untreated teases, and primary neutrophils are generally not amenable to other controls). modes of inhibition such as gene silencing because they are termi- nally differentiated and short-lived (Rock et al., 1994). The analysis in the presence of other activating agents such as fMLP and antag- of adhesion at early time points (30–45 min) is also critical because onized by inhibitors of calcium flux (Yuo et al., 1989). Therefore, of the known involvement of TNF- in regulating NF-kB activity we tested whether or not calpain inhibitors affect TNF- -mediated (Read et al., 1995). Previous studies have reported that ALLM and release of reactive oxygen species (Fig. 6). Neutrophils were stim- ALLN have different effects on adhesion mediated by NF- B due to ulated with TNF- for 30 min in the presence or absence of calpain their different inhibitory potencies with regard to the proteasome inhibitors and generation of hydrogen peroxide in the media (Read et al., 1995). In our studies, ALLM and ALLN affected adhe- was quantified. Following TNF- stimulation of fMLP-treated neu- sion similarly in TNF- -treated neutrophils, suggesting the effects trophils, levels of hydrogen peroxide were increased approximately on adhesion are due to calpain inhibition rather than non-specific effects on the proteasome. It has been shown by us and others that TNF- stimulation induces a flattened, non-polarized morphology characterized by the formation of vinculin-containing focal complexes (Fuortes et al., 1999; Lokuta and Huttenlocher, 2005). Here, we demonstrate that treatment of neutrophils with TNF- in the presence of cal- pain inhibitors induces a polarized morphology with a concomitant relocalization of actin to the leading edge, tubulin orientation to the rear, and reduction of vinculin-containing focal contacts. Focal complexes form a platform for NADPH oxidase-mediated gener- ation of reactive oxygen species (Williams and Solomkin, 1999). We also find that calpain inhibition impairs TNF- -mediated neu- trophil oxidative burst. Our data are in agreement with work from others showing that both adhesion (Nathan, 1987) and calcium influx (Brechard and Tschirhart, 2008) are required for TNF- - induced oxidative burst. The disruption in the ability of TNF- to induce stable focal complexes may well underlie the effect that cal- Fig. 6. Calpain inhibition prevents TNF- -mediated oxidative burst. Human periph- pain inhibition has on the generation of oxidative burst after TNF- eral blood neutrophils were treated for 30 min with TNF- (250 ng/mL) in the treatment. presence or absence of calpain inhibitors ALLN or ALLM (50 g/mL), Z-LLY-FMK We have demonstrated that calpain inhibition induces random (45 M), or PD150606 (1 M). H2 O2 production was quantified relative to unstim- ulated controls (mean ± SEM, n = 4 or 5, p < 0.05 by one-way ANOVA, Tukey post hoc migration in TNF- -treated arrested neutrophils. While all four analysis indicates significant difference relative to control (*) or fMLP TNF (#)). calpain inhibitors induced this effect, the magnitude was larger
  • A.J. Wiemer et al. / Molecular Immunology 47 (2010) 894–902 901 inflammation. The data presented here could suggest a mecha- nism for the previous finding that calpain inhibition in models of arthritis prevents neutrophil accumulation in sites of inflamma- tion (Cuzzocrea et al., 2000). Ultimately, data from more complex in vivo models would further clarify the precise physiological roles of calpains in mediating TNF- signaling. Acknowledgements This work was supported by NIH NIAID R01 to A.H. [AI068062]. Postdoctoral support was provided to A.J.W. by the University of Wisconsin Institute on Aging Training Grant (NIH [T32AG000213- 17], Sanjay Asthana, P.I.). We would like to thank Gary Bokoch for useful discussions. Fig. 7. TNF- and calpain regulate integrin activation in human neutrophils. Binding of TNF- to its receptor initiates a signaling cascade that increases -integrin- References mediated adhesion, promoting neutrophil arrest. Neutrophil arrest requires p38 activation (Lokuta and Huttenlocher, 2005). Calpain inhibition prevents calpain- mediated proteolysis of the focal contact substrates talin, paxillin, and integrins, Aggarwal, B.B., 2003. Signalling pathways of the TNF superfamily: a double-edged sword. Nat. Rev. Immunol. 3, 745–756. which may alter neutrophil adhesion. Aloisi, F., Pujol-Borrell, R., 2006. Lymphoid neogenesis in chronic inflammatory dis- eases. Nat. Rev. Immunol. 6, 205–217. Brechard, S., Tschirhart, E.J., 2008. Regulation of superoxide production in neu- trophils: role of calcium influx. J. Leukoc. Biol. 84, 1223–1237. with ALLN and ALLM than for LLY-FMK and PD150606. This is Chen, H.C., Wang, C.J., Chou, C.L., Lin, S.M., Huang, C.D., Lin, T.Y., Wang, C.H., Lin, not surprising because ALLN and ALLM are generally regarded as H.C., Yu, C.T., Kuo, H.P., Liu, C.Y., 2006. Tumor necrosis factor-alpha induces more potent inhibitors. These findings suggest that calpain activ- caspase-independent cell death in human neutrophils via reactive oxidants and associated with calpain activity. J. Biomed. Sci. 13, 261–273. ity is required for TNF- -induced neutrophil arrest. We speculate Cuzzocrea, S., McDonald, M.C., Mazzon, E., Siriwardena, D., Serraino, I., Dugo, L., based on these data and others in the literature that calpain may Britti, D., Mazzullo, G., Caputi, A.P., Thiemermann, C., 2000. Calpain inhibitor I act to prevent protrusive behavior and as a result of the inhibi- reduces the development of acute and chronic inflammation. Am. J. Pathol. 157, 2065–2079. tion of calpain, arrested neutrophils exhibit decreased adhesion Diamond, M.S., Springer, T.A., 1993. A subpopulation of Mac-1 (CD11b/CD18) and enhanced chemokinesis. The effects of calpain inhibition are molecules mediates neutrophil adhesion to ICAM-1 and fibrinogen. J. Cell Biol. similar to the phenotype observed with inhibitors of microtubules 120, 545–556. such as colchicine, which increase random migration but decrease Diaz, F., Bourguignon, L.Y.W., 2000. Selective down-regulation of IP3 receptor sub- types by caspases and calpain during TNF alpha-induced apoptosis of human directional chemotaxis (Xu et al., 2005). T-lymphoma cells. Cell Calcium 27, 315–328. In primary human neutrophils TNF- stimulation promotes Drost, E.M., MacNee, W., 2002. Potential role of IL-8, platelet-activating factor and phosphorylation of p38 and ERK, the former of which is required TNF-alpha in the sequestration of neutrophils in the lung: effects on neutrophil deformability, adhesion receptor expression, and chemotaxis. Eur. J. Immunol. for TNF-mediated arrest (Lokuta and Huttenlocher, 2005). Calpain 32, 393–403. inhibition did not significantly change the amount of phosphory- Franco, S.J., Huttenlocher, A., 2005. Regulating cell migration: calpains make the cut. lated p38 or ERK induced by TNF- . This suggests that the role J. Cell Sci. 118, 3829–3838. Fuortes, M., Melchior, M., Han, H., Lyon, G.J., Nathan, C., 1999. Role of the tyrosine calpain plays in neutrophil adhesion regulation is independent of kinase pyk2 in the integrin-dependent activation of human neutrophils by TNF. altered MAPK signaling. We hypothesize that calpain activity acts J. Clin. Invest. 104, 327–335. downstream of the TNF receptor and is required for neutrophil Gillitzer, R., Goebeler, M., 2001. Chemokines in cutaneous wound healing. J. Leukoc. Biol. 69, 513–521. adhesion, arrest, and oxidative burst (Fig. 7). However, it is unlikely Granger, D., Kubes, P., 1994. The microcirculation and inflammation: modulation of that calpain is acting via a p38 MAPK or ERK pathway in order to leukocyte-endothelial cell adhesion. J. Leukoc. Biol. 55, 662–675. carry out these functions. A more plausible scenario may involve Hendey, B., Lawson, M., Marcantonio, E., Maxfield, F., 1996. Intracellular calcium and calcineurin regulate neutrophil motility on vitronectin through a receptor calpain regulation of focal complex formation via cleavage of its identified by antibodies to integrins alphav and beta3. Blood 87, 2038–2048. known substrates such as talin. Huang, Y., Wang, K.K., 2001. The calpain family and human disease. Trends Mol. There is now an accumulating body of evidence describing Med. 7, 355–362. calpain-mediated effects of TNF- signaling in neutrophils (Vanags Huttenlocher, A., Palecek, S.P., Lu, Q., Zhang, W.L., Mellgren, R.L., Lauffenburger, D.A., Ginsberg, M.H., Horwitz, A.F., 1997. Regulation of cell migration by the calcium- et al., 1996; Diaz and Bourguignon, 2000; Chen et al., 2006). Under dependent protease calpain. J. Biol. Chem. 272, 32719–32722. some circumstances TNF- promotes survival of neutrophils, how- Katsube, M., Kato, T., Kitagawa, M., Noma, H., Fujita, H., Kitagawa, S., 2008. Calpain- ever TNF- signaling also has been linked to neutrophil apoptosis mediated regulation of the distinct signaling pathways and cell migration in human neutrophils. J. Leukoc. Biol. 84, 255–263. (Chen et al., 2006). TNF- -treatment alone induces neutrophil Kulkarni, S., Saido, T.C., Suzuki, K., Fox, J.E.B., 1999. Calpain mediates integrin- arrest, but was recently shown to enhance neutrophil migration induced signaling at a point upstream of Rho family members. J. Biol. Chem. in response to certain chemoattractants (Montecucco et al., 2008). 274, 21265–21275. Locksley, R.M., Killeen, N., Lenardo, M.J., 2001. The TNF and TNF receptor superfam- While TNF- signaling during migration, adhesion and apoptosis ilies: integrating mammalian biology. Cell. 104, 487–501. is complex, all of these important cellular processes appear to be Lokuta, M.A., Huttenlocher, A., 2005. TNF-alpha promotes a stop signal that inhibits dependent upon calpain activity and altered by calpain inhibitors. neutrophil polarization and migration via a p38 MAPK pathway. J. Leukoc. Biol. 78, 210–219. Our work strengthens this accumulating body of knowledge by Lokuta, M.A., Nuzzi, P.A., Huttenlocher, A., 2003. Calpain regulates neutrophil showing that calpain inhibition impairs TNF- -mediated adhesion, chemotaxis. Proc. Natl. Acad. Sci. U.S.A. 100, 4006–4011. stop signal and oxidative burst. Lokuta, M.A., Nuzzi, P.A., Huttenlocher, A., 2007. Analysis of neutrophil polarization and chemotaxis. Methods Mol. Biol. 412, 211–229. It is unknown whether altering neutrophil adhesion and migra- Mackay, C.R., 2008. Moving targets: cell migration inhibitors as new anti- tion would be an effective means to treat inflammatory disorders. inflammatory therapies. Nat. Immunol. 9, 988–998. Neutrophil retention in inflamed tissues is enhanced by TNF- and Mayadas, T.N., Cullere, X., 2005. Neutrophil [beta]2 integrins: moderators of life or exacerbates various disease states. Calpain inhibition, by enhanc- death decisions. Trends Immunol. 26, 388–395. McLeish, K.R., Knall, C., Ward, R.A., Gerwins, P., Coxon, P.Y., Klein, J.B., Johnson, G.L., ing migration in TNF- stimulated neutrophils and preventing their 1998. Activation of mitogen-activated protein kinase cascades during priming accumulation in inflamed sites, may be one approach to reduce of human neutrophils by TNF-alpha and GM-CSF. J. Leukoc. Biol. 64, 537–545.
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