A.J. Wiemer et al. / Molecular Immunology 47 (2010) 894–902 895
arrest (Lokuta and Huttenlocher, 2005; Mayadas and Cullere, 2.2. Neutrophil isolation
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
activity in an animal model of collagen-induced arthritis blocked
neutrophil recruitment and prevented inﬂammation (Cuzzocrea et
2.4. Adhesion assay
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.
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.
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
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
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
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
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.
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
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.
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
Fig. 7. TNF- and calpain regulate integrin activation in human neutrophils. Binding
of TNF- to its receptor initiates a signaling cascade that increases -integrin-
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
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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,
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.
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such as colchicine, which increase random migration but decrease Diaz, F., Bourguignon, L.Y.W., 2000. Selective down-regulation of IP3 receptor sub-
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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.
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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.
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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
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