Cyclophilin A cooperates with MIP-2 to augment neutrophil migration

Article (PDF Available)inJournal of Inflammation Research 4(1):93-104 · June 2011with43 Reads
DOI: 10.2147/JIR.S20733 · Source: PubMed
Abstract
Chemokines contribute to inflammatory responses by inducing leukocyte migration and extravasation. In addition, chemoattractants other than classical chemokines can also be present. Many chemokines have been demonstrated to cooperate, leading to an augmentation in leukocyte recruitment and providing a potential role for the presence of multiple chemoattractants. Extracellular cyclophilins are a group of alternative chemotactic factors, which can be highly elevated during various inflammatory responses and, as we have previously shown, can contribute significantly to neutrophil recruitment in an animal model of acute lung inflammation. In the current studies we investigated whether the most abundant extracellular cyclophilin, CypA, has the capacity to function in partnership with 2 classical chemokines known to be secreted in the same model, macrophage inflammatory protein (MIP)-2/CXCL2 and keratinocyte chemoattractant (KC)/CXCL1. Neutrophil migration in response to combinations of CypA and MIP-2 or CypA and KC was measured by in vitro chemotaxis assays. Biochemical responses of neutrophils incubated with the combinations of chemoattractants were determined by changes in chemokine receptor internalization and actin polymerization measured by flow cytometry, and changes in intracellular calcium mobilization measured with a calcium sensitive fluorochrome. A combination of CypA and MIP-2, but not KC, augmented neutrophil migration. Based on the level of augmentation, the cooperation between CypA and MIP-2 appeared to be synergistic. Evidence that CypA and MIP-2 cooperate at the biochemical level was demonstrated by increases in receptor internalization, calcium mobilization, and actin polymerization. These findings provide evidence for the capacity of extracellular cyclophilins to interact with classical chemokines, resulting in greater and more efficient leukocyte recruitment.
© 2011 Heine et al, publisher and licensee Dove Medical Press Ltd. This is an Open Access article
which permits unrestricted noncommercial use, provided the original work is properly cited.

Journal of Inflammation Research Dovepress
submit your manuscript | www.dovepress.com
Dovepress
93

open access to scientific and medical research
Open Access Full Text Article
DOI: 10.2147/JIR.S20733

neutrophil migration

1
Denise Olive
1

2
Philip M Murphy
2
Michael I Bukrinsky
1

1
1
Department of Microbiology,
Immunology and Tropical Medicine,


2
Laboratory

Institute of Allergy and Infectious






Tel +
Fax +
m
Background: Chemokines contribute to inflammatory responses by inducing leukocyte
migration and extravasation. In addition, chemoattractants other than classical chemokines can
also be present. Many chemokines have been demonstrated to cooperate, leading to an aug-
mentation in leukocyte recruitment and providing a potential role for the presence of multiple
chemoattractants. Extracellular cyclophilins are a group of alternative chemotactic factors,
which can be highly elevated during various inflammatory responses and, as we have previously
shown, can contribute significantly to neutrophil recruitment in an animal model of acute lung
inflammation. In the current studies we investigated whether the most abundant extracellular
cyclophilin, CypA, has the capacity to function in partnership with 2 classical chemokines
known to be secreted in the same model, macrophage inflammatory protein (MIP)-2/CXCL2
and keratinocyte chemoattractant (KC)/CXCL1.
Methods: Neutrophil migration in response to combinations of CypA and MIP-2 or CypA
and KC was measured by in vitro chemotaxis assays. Biochemical responses of neutrophils
incubated with the combinations of chemoattractants were determined by changes in chemokine
receptor internalization and actin polymerization measured by flow cytometry, and changes in
intracellular calcium mobilization measured with a calcium sensitive fluorochrome.
Results: A combination of CypA and MIP-2, but not KC, augmented neutrophil migration.
Based on the level of augmentation, the cooperation between CypA and MIP-2 appeared to be
synergistic. Evidence that CypA and MIP-2 cooperate at the biochemical level was demonstrated
by increases in receptor internalization, calcium mobilization, and actin polymerization.
Conclusion: These findings provide evidence for the capacity of extracellular cyclophi-
lins to interact with classical chemokines, resulting in greater and more efficient leukocyte
recruitment.
Keywords: chemokine, chemotaxis, inflammation
Introduction
Neutrophils are integral leukocytes in the monitoring of the immune system and the host
response to inflammation. The main regulators of leukocyte trafficking are chemokines,
a family of chemoattracting cytokines. Chemokines induce cell migration by binding
to and signaling through G-protein-coupled receptors (GPCRs) on their target cells.
While GPCRs for chemokines are restricted to interaction with specific families of
this class of chemoattractants, notably CC versus CXC, many can act as receptors for
several different members within a family. The fact that inflammatory processes are
usually associated with the production of many different chemokines, some of which
target the same leukocyte subset or even the same specific receptors on target cells,
suggests a significant overlap and/or redundancy in function by chemokines.
Number of times this article has been viewed
This article was published in the following Dove Press journal:
Journal of Inammation Research
2 June 2011

submit your manuscript | www.dovepress.com
Dovepress
Dovepress
94
Heine et al
To add to the already complex mix of multiple chemokines
is the finding that additional factors with chemoattracting
properties can also be present during inflammatory responses,
for example, cyclophilins, a family of intracellular proteins
that are ubiquitously expressed in all organisms and in all
human tissues. Cyclophilins can be secreted by different cell
types in response to inflammatory stimuli such as LPS
1,2
or
reactive oxygen radicals,
3
thereby generating extracellular
pools of these proteins. Indeed, high levels of extracellular
cyclophilins have been reported in several inflammatory
diseases, both in humans
2,4,5
and mice.
6–8
We have previously
proposed that extracellular cyclophilins might play a role in
inflammatory processes by contributing to the recruitment
of leukocytes to sites of ongoing inflammation.
9
Indeed,
using 3 different animal models of inflammatory disease,
our laboratory demonstrated that inhibiting the chemotactic
function of cyclophilins using either a nonimmunosuppres-
sive analog of CsA
6
or an antibody to the principal signaling
receptor for extracellular cyclophilins A and B, CD147,
6–8
can
significantly reduce leukocyte recruitment in vivo.
Studies in which classical chemokines are inhibited also
demonstrate significant reductions in leukocyte recruitment.
In the case of neutrophils, for example, mice given anti-
MIP-2 antibody showed a significantly reduced neutrophil
influx into lungs challenged with bacteria
10
and also into
LPS-treated air pouches.
11
Treatment with a blocking anti-
body against the CXC chemokine, LIX (CXCL5), signifi-
cantly decreased neutrophil migration to airways and lungs
of mice in an LPS model of acute lung injury.
12
Inhibiting
the signaling function of another chemokine, CCL2, using
mice lacking CCR2 receptors, resulted in a greater than
90% decrease in alveolar neutrophil influx.
13
Given that
the total contribution of all reported chemokines, as well as
non-classical chemokines, produced during an inflammatory
response cannot be greater than 100%, the possibility that
some of these chemoattracting factors might cooperate has
been explored from different mechanistic perspectives.
One such mechanism is chemokine synergy in which
the total chemotactic function that is mediated by combin-
ing 2 chemokines is greater than their additive individual
functions. Therefore, when one chemokine in a coopera-
tive partnership is inhibited, the function of the second
chemokine is indirectly impacted, resulting in a greater total
reduction in cell recruitment and a potential explanation for
the apparent .100% inhibition in leukocyte recruitment
when individual effects are added together. Many different
chemokines have been shown to work in partnership both in
vitro and in vivo, with the potential to amplify inflammatory
responses (reviewed in
14
). Following from our long-standing
interest in the contribution of extracellular cyclophilins as
chemoattractants during inflammatory responses, we inves-
tigated in the current studies whether cyclophilin A (CypA),
the most abundant member of the cyclophilin family,
15
might have a similar capacity to cooperate with classical
chemokines to mediate augmented leukocyte migration. In
previous studies we showed that CypA is a potent inducer
of neutrophil migration in vitro and that high levels of CypA
are present in the airways of mice with ongoing pulmonary
neutrophilia induced by intranasal administration of LPS.
6
We therefore examined whether combining CypA with
other known neutrophil-attracting chemokines would lead
to an increase in neutrophil recruitment, and the impact of
these combinations on biochemical events associated with
cell migration.
In this report we show that CypA is able to cooperate with
MIP-2, but not KC, to induce a synergistic augmentation in
neutrophil migration in vitro. Analysis of various parameters
associated with cell migration demonstrated that neutrophils
stimulated with a combination of CypA and MIP-2 showed
greater increases in GPCR (CXCR2) internalization, intracel-
lular calcium mobilization, and actin polymerization, relative
to cells stimulated with the individual chemoattractants.
These additional increases were not observed in neutrophils
stimulated with combinations of CypA and KC. Taken
together, these findings provide evidence that CypA has the
capacity to interact with selective classical chemokines to
mediate an augmented neutrophil migration that is associated
with enhanced biochemical events.
Material and methods
Animals
Female C57BL/6 mice, at least 6 weeks of age, were used for
these studies and were purchased from the National Cancer
Institute (Bethesda, MD). Blood, or bone marrow, was pooled
from 2 to 5 age-matched animals for each experiment. All
studies were reviewed and approved by the Institutional
Animal Care and Use committee at The George Washington
University.

PE-conjugated anti-mouse Gr-1 was purchased from BD
Biosciences (San Jose, CA) and APC-conjugated anti-mouse
Gr-1 was purchased from eBioscience (San Diego, CA). PE-
conjugated anti-mouse CXCR2 and rat IgG
2A
isotype were
purchased from R&D Systems (Minneapolis, MN). FITC-
conjugated rat anti-mouse CD147 monoclonal antibody and

submit your manuscript | www.dovepress.com
Dovepress
Dovepress
95

IgG1 isotype control were purchased from Abcam (Cambridge,
MA). FITC-labeled phalloidin for actin polymerization stud-
ies was purchased from Sigma-Aldrich (St. Louis, MO).
Anti-human cyclophilin A mAb (cross-reacts with mouse
cyclophilin A) was obtained from US Biological (Swampscott,
MA), and HRP-conjugated donkey anti-rabbit secondary Ab
was purchased from Amersham Biosciences (Piscataway,
NJ). fMLP, and BSA Fraction V were obtained from Sigma-
Aldrich. Human recombinant CypA, which differs from mouse
CypA by only one amino acid residue, was purchased from
Calbiochem (San Diego, CA). Recombinant mouse MIP-2
and KC were purchased from PeproTech (Rocky Hill, NJ).
FLIPR calcium 3 assay kit component A fluorescent dye for
calcium measurements was purchased from Molecular Devices
(Sunnyvale, CA) and dATP from Invitrogen (Carlsbad, CA).

For chemotaxis assays: mouse peripheral blood was col-
lected by cardiac puncture. The collected blood was treated
with RBC lysis buffer and the neutrophils were then isolated
using a MACS Gr-1 positive selection kit (Miltenyi Biotech,
Aurora, CA). For actin polymerization and intracellular cal-
cium experiments: bone marrow-derived neutrophils were
obtained by flushing tibias and femurs of C57BL/6 mice with
HBSS. The isolated bone marrow was washed with HBSS,
centrifuged at 300 g for 12 minutes, and resuspended in
6 mL HBSS. The cells were layered on top of a Histopaque
1077/1119 gradient (3 mL Histopaque 1119 overlayed with
3 mL Histopaque 1077) and centrifuged at room temperature
for 30 minutes at 700 g. The resulting granulocyte layer
was removed, washed, and then used for phalloidin stain-
ing or calcium assays. For receptor internalization studies:
mouse peripheral blood was collected by cardiac puncture.
The collected blood was treated with RBC lysis buffer
and the neutrophils were then isolated using an EasySep
Mouse Neutrophil Enrichment Kit (Stem Cell Technologies
Vancouver, BC, Cananda). Purity for all enriched populations
was established to be 80 to 90% by FACS analysis.

Chemotaxis assays were set up using 48-well modified
Boyden chambers (Neuro Probe Inc., Gaithersburg, MD)
with the 2 compartments separated by a 5-µm polycarbonate
membrane (Neuro Probe). Neutrophils (10
4
cells/well) in
RPMI 1640 culture medium supplemented with 1% BSA
were added to the upper chamber wells, while media con-
taining different doses and combinations of chemoattractants
(shown in Figures) were added to the lower compartments.
In some studies purified mouse neutrophils were preincubated
in medium alone (RPMI 1640 + 1% BSA fraction V), or
medium containing doses of recombinant CypA or MIP-2
(as indicated in figure legends) for 30 minutes before being
washed in PBS, resuspended in fresh medium, and applied to
the upper wells of the chamber. A dose of 10
-7
M fMLP was
used as a positive control for neutrophil migration. The cham-
bers were incubated at 37°C in 5% CO
2
for 50 minutes, after
which the chamber membrane was removed, non-migrating
cells scraped off, and the membrane stained with Wright-
Giemsa (Camco, Fort Lauderdale, FL). A chemotactic
index was generated for each well by dividing the number
of cells counted for that well by the number of cells counted
in media wells.

Purified mouse neutrophils were serum starved in RPMI 1640
without BSA for 30 minutes at room temperature. This short
starvation step was included to reduce background staining
while maintaining maximal cell viability. The cells were
then supplemented with an equivalent volume of RMPI 1640
containing 2% BSA fraction V in preparation for stimulation.
1 × 10
6
neutrophils in 200 µL volume medium (RMPI + 1%
BSA fraction V) were set up in a 96-well tissue culture plate.
Cells were incubated in medium alone, or with recombinant
CypA, MIP-2, or KC, as well as combinations of the various
chemoattractants (as indicated in figure legends) for 5 min-
utes at 37°C in 5% CO
2
. (Five minutes was chosen based on
initial kinetics studies showing optimal changes at this time
point). The cells were then removed from wells, washed with
PBS, and stained with APC-Gr-1 for gating on neutrophils.
The cells were also stained with either FITC anti-CD147,
FITC IgG1 isotype control, PE-CXCR2, or PE IgG
2A
control,
and expression of CD147 and CXCR2 receptors on Gr-1
positive cells was determined by flow cytometry.
Intracellular calcium measurements
Intracellular calcium measurements were conducted with
bone marrow neutrophils using a Benchtop Scanning
Fluorometer and Integrated Fluid Transfer Workstation
(FlexStation; Molecular Devices, Sunnyvale, CA). A total of
1 × 10
6
cells in 100 µL HBSS + 20 mM HEPES buffer was
loaded into a 96-well plate together with 100 µL of FLIPR
calcium 3 assay kit component A fluorescent dye (Molecular
Devices), and then incubated for 30 min at 37°C. A separate
plate containing various doses of chemoattractants (shown in
figure legends) was set up. Both plates were then loaded into
the FlexStation for automated addition of chemoattractants

submit your manuscript | www.dovepress.com
Dovepress
Dovepress
96
Heine et al
to the fluorescently labeled cells. Baseline fluorescence was
established before the chemoattractants were added to cells
at 20 seconds. Changes in intracellular calcium concentra-
tions in response to added chemoattractants were recorded
as relative fluorescence units over time. dATP (100 µM)
was added to all groups of cells at 120 seconds as a positive
control for equal capacity to flux calcium.
Actin polymerization
Bone marrow neutrophils were serum starved in 500 µL
RPMI 1640 without BSA for 30 minutes at room temperature
and then supplemented with 500 µL RMPI 1640 containing
2% BSA fraction V in preparation for stimulation. 5 × 10
6
neutrophils (now in RMPI 1640 with 1% BSA medium)
were incubated at 37°C in 5% CO
2
for 5 minutes in medium
alone, fMLP as a positive control, CypA, MIP-2, KC alone
or various combinations, at doses indicated in figure legends.
(Five minutes was chosen based on initial kinetics studies
showing optimal changes at this time point). The cells
were then immediately fixed by adding 3.7% formalin for
10 minutes at room temperature. Samples were centrifuged
for 1 minute at 1400 rpm and supernatants discarded. 100 µL
cold 0.1% Triton X-100 was added to each sample and
incubated on ice for 30 minutes to permeabilize the cells.
Samples were washed with FACS buffer and stained with
6 µg/mL FITC-conjugated phalloidin in FACS buffer at
room temperature for 40 minutes. Actin polymerization was
determined by the level of phalloidin expression, as measured
by flow cytometry.

Data are summarized as mean ± SE. Statistical analysis of
the results was performed using an unpaired Student t test,
with P , 0.05 considered significant.

One supplemental figure is provided. Supplemental
Figure 1 shows in vitro neutrophil migration in response
to different doses of CypA, MIP-2, and KC. These titra-
tion curves were used to select suboptimal doses for each
chemoattractant for the combination studies.
Results


In previous studies we demonstrated the capacity of CypA
to induce in vitro migration of human
16
and mouse
6
neutro-
phils. In the current studies we investigated whether CypA
might have the capacity to interact with classical chemokines
known to be potent inducers of neutrophil migration, notably
MIP-2 and KC, to mediate enhanced migration. Based on
the findings of others that functional interactions are most
likely to occur when suboptimal doses of chemokines are
combined,
17
we initially established individual dose response
curves for all 3 chemoattractants (Supplemental Figure 1)
and then selected doses in the lower range for testing in our
combination assays. The data in Figure 1 show results from
experiments in which neutrophils were set up with a fixed
dose of MIP-2 or KC plus varying doses of CypA. Our find-
ings demonstrate that combining CypA with MIP-2 induces a
significant augmentation in neutrophil migration (Figure 1A).
Interestingly, this augmentation was greater than the additive
migration induced by MIP-2 alone plus CypA alone, sug-
gesting that the 2 chemokines are interacting in a synergistic
partnership. No such enhancement in neutrophil migration
was observed using various combinations of KC and CypA
15.0
12.5
10.0
7.5
5.0
2.5
1.0
4.0
3.5
3.0
2.0
1.5
1.0
2.5
Chemotactic index
A
fMLP MIP-2
CypA
-
-
10 10 10
2.5 55
10
10
10
20
ng/mL
***
*
Chemotactic index
B
fMLP KC
CypA
-
-
20 20 20 20 20
2.5 551020
ng/mL
Figure 1 


      
group by dividing the number of migrated cells in test wells by the number of cells
that migrated to medium alone. A) Mean +

B) Mean +

Notes:t= 6 wells per
group. *P , 0.05 and ***P , 0.001. These data are representative of .
experiments.
Abbreviations:

submit your manuscript | www.dovepress.com
Dovepress
Dovepress
97

(Figure 1B). Taken together, these findings demonstrate that
the presence of CypA can significantly increase the capacity
of MIP-2 to induce neutrophil migration and that this effect
is more than just additive.

concurrently for cooperation to occur
We next examined whether the augmented neutrophil
migration observed when CypA and MIP-2 are combined
requires that the 2 chemoattractants be present concurrently,
or whether the same effect could be mediated by sequential
exposure. Thus, in vitro chemotaxis assays were conducted
in which neutrophils were preincubated with different doses
of one chemoattractant, washed, and then tested for their
migratory response to the second chemoattractant. As shown
in Figure 2A, neutrophils preincubated with CypA did not
respond either additively or synergistically upon subsequent
stimulation with MIP-2. Likewise, neutrophils preincubated
with MIP-2 did not migrate with either an additive or syn-
ergistic response to CypA (Figure 2B). Importantly, in both
experiments the concurrent exposure to MIP-2 and CypA led
to a greater than additive increase in neutrophil migration,
demonstrating the reproducibility of the synergistic interac-
tion when the 2 chemoattractants are present together.


Ligand binding to chemokine receptors triggers a cascade
of signaling events that ultimately result in cell polariza-
tion and chemotaxis, as well as receptor internalization.
18
Increases in receptor internalization are often used as an
indicator of increased receptor engagement and/or receptor
signaling. In the current studies we investigated the impact of
co-incubation with CypA and MIP-2, relative to incubation
with the individual chemoattractants, on the internalization
of CD147 (receptor for CypA
19
) and CXCR2 (receptor for
MIP-2) on neutrophils. It should be noted that the same
low doses of CypA and MIP-2, which demonstrated syner-
gistic responses (Figures 1 and 2), were used in the current
studies. Combinations of CypA and KC were also included
as a comparison for effects mediated by noncooperating
chemoattractants. Figures 3A, 3B, and 3E show that CD147
expression was not significantly changed (based on MFI units)
after incubation with CypA, MIP-2, or KC alone, or with any
of the chemoattractant combinations. In contrast, stimulation
with a combination of CypA and MIP-2 resulted in a signifi-
cantly greater CXCR2 receptor internalization (average of
26% reduction in expression compared with medium alone,
based on MFI units), relative to that induced by MIP-2 alone
(13% reduction) (Figures 3C and 3F). Adding CypA to KC
did not significantly augment the degree of CXCR2 inter-
nalization mediated by KC alone (11% and 16% reductions,
respectively) (Figures 3D and 3F). Taken together, these data
demonstrate a selective increase in CXCR2 internalization
when CypA and MIP-2 are present in combination, suggesting
enhanced receptor engagement and/or signaling.

intracellular calcium mobilization
Intracellular calcium release is triggered upon GPCR binding
and activation, and therefore increases in intracellular cal-
cium levels are often used as a readout of GPCR activation.
Similarly, intracellular calcium release is triggered by CD147
engagement by cyclophilins. Cooperating chemokines have
been shown to induce enhanced calcium flux when added in
combination.
18,20,21
Having demonstrated increased receptor
internalization in the presence of CypA and MIP-2, we next
conducted calcium flux assays to provide further support
for additive signaling between CypA and MIP-2. Calcium
release in response to the individual chemoattractants, or
their combination, was measured in neutrophils in real
time using a Molecular Devices FlexStation. As shown
in Figure 4, the greatest increase in intracellular calcium
levels was observed when CypA and MIP-2 were present
in combination. Moreover, the increase in calcium flux
mediated by the combination appeared to be more rapid and
more prolonged, compared with that mediated by MIP-2 and
CypA alone. These findings provide additional evidence
for the capacity of a MIP-2/CypA combination to mediate
augmented signaling.

increased actin polymerization
F-actin polymerization occurs at the leading edge of neutro-
phils in response to chemokine-mediated GPCR stimulation.
Cooperating chemokines have been shown to trigger
increased actin polymerization when present in combination
compared with individual chemokines,
22
providing a potential
mechanism for enhanced leukocyte migration by synergiz-
ing chemoattractants. In the current studies neutrophils were
stimulated with CypA, MIP-2, or KC alone, or with combina-
tions of CypA and MIP-2 or CypA and KC, followed by stain-
ing with FITC-conjugated phalloidin to measure the extent of
actin polymerization. Phalloidin expression was significantly
increased in neutrophils incubated with a combination of
CypA and MIP-2 (average of 33% increase compared with

submit your manuscript | www.dovepress.com
Dovepress
Dovepress
98
Heine et al
medium alone, based on MFI units), compared with those
incubated with individual chemoattractants (MIP-2 = 5%
decrease and CypA = 6% increase) (Figures 5A and 5C). No
such changes in phalloidin expression were mediated when
CypA and KC were combined (KC alone = 23% increase
versus KC + CypA = 14% increase) (Figures 5B and 5C).
Discussion
Neutrophil migration toward sites of infection is integral
in the innate immune response. Chemokines secreted by
resident cells in response to inflammatory stimuli establish a
gradient that induces neutrophils to extravasate and migrate to
infected tissues. Although beneficial for pathogen clearance,
the neutrophilia that ensues during inflammatory responses
often results in extensive bystander tissue damage. Neutrophil
accumulation and activation within tissues contributes
significantly to the pathology of both autoimmune and non-
autoimmune inflammatory conditions (reviewed in
23
) and can
also promote the onset of carcinogenic events (reviewed in
24
).
Therefore, understanding how neutrophil recruitment is regu-
lated, including the factors that contribute to this regulation,
has important therapeutic implications for a wide variety of
diseases associated with tissue injury.
Many chemokines are secreted during inflammation,
suggesting either a high degree of redundancy or an overlap
in function. Interestingly, several chemokines have been
9
7
5
3
1
0
10
5
0
0
0
0
10
0
10
0
0
5
10
CypA (ng/mL)
MIP-2 (ng/mL)
Preincubation
(30 minutes)
Medium 5 ng/mL
CypA
50 ng/mL
CypA
Chemotactic index
NS
NS
*
*
A
1
2
3
4
5
6
7
0
10
5
0
0
0
5
0
5
0
0
0
5
10
CypA (ng/mL)
MIP-2 (ng/mL)
Chemotactic index
NS
NS
*
*
Preincubation
(30 minutes)
Medium 10 ng/mL
MIP-2
100 ng/mL
MIP-2
B
Figure 2                        



of migrated cells in test wells by the number of cells preincubated in medium that migrated to medium alone. A) Mean +
B) Mean +

Notes:t= 6 wells per group. *P , 0.05. These data are representative of .
Abbreviations:

submit your manuscript | www.dovepress.com
Dovepress
Dovepress
99

10
4
10
3
10
2
10
1
0
Line
Group
MFI
Isotype
CypA + MIP-2
MIP-2
CypA
Medium
46.6
432
414
426
421
Relative cell number
CD147
A
10
4
10
3
10
2
10
1
0
Line
Group
MFI
Isotype
CypA + KC
KC
CypA
Medium
39.4
362
375
426
421
Relative cell number
CD147
B
10
4
10
3
10
4
10
2
10
1
0
Line
Group
MFI
Isotype
CypA + MIP-2
MIP-2
CypA
Medium
144
969
1135
1566
1373
Relative cell number
CXCR2
C
10
4
10
3
10
2
10
1
0
Line
Group
MFI
Isotype
CypA + KC
KC
CypA
Medium
144
1107
1072
1156
1373
Relative cell number
CXCR2
D
50
40
30
20
10
0
−10
CypA MIP-2 KCCypA
+
MIP-2
CypA
+
KC
% decrease in CD147 MFI
E
CypA
+
KC
50
40
30
20
10
0
−10
CypA MIP-2 KCCypA
+
MIP-2
% decrease in CXCR2 MFI
F
*
**
Figure 3 

   A        
+B+C
+D
+
EF
Abbreviations:
shown to work in partnership with the potential to amplify
inflammatory responses. For example, studies with the
CC plasma chemokine, Regakine-1, showed a synergis-
tic enhancement in neutrophil migration when combined
with IL-8 (CXCL8), GCP-2 (CXCL6), MCP-3 (CCL7),
25
or with NAP-2
26
in vitro, and when co-administered with
GCP-2 in vivo.
27
Interestingly, Regakine-1 was also shown
to synergize with a chemotactic anaphylatoxin, C5a,
26
sug-
gesting that these types of interactions are not restricted
to classical chemokines. Other studies have found that

submit your manuscript | www.dovepress.com
Dovepress
Dovepress
100
Heine et al
IL-8 cooperates with GM-CSF in the female reproductive
tract, resulting in enhanced neutrophil chemotaxis.
28
This
enhanced recruitment was inhibited by up to 95% when the
2 chemokines were neutralized in combination.
28
RANTES
(CCL5) and MCP-1 (CCL2) were shown to work together to
enhance the migration of PBMCs in vitro
18
and the in vivo
co-injection of RANTES and IP-10 (CXCL10) was reported
to augment T lymphocyte recruitment.
29
IP-10 has also been
shown to increase the migration of Th1 and Th2 clones when
combined with MDC (CCL22) in vitro.
22
Extracellular cyclo-
philins are another family of proteins with chemoattracting
properties that are elevated during inflammatory responses.
Our laboratory has previously demonstrated that extracel-
lular cyclophilins contribute significantly to neutrophil
recruitment in a mouse model of LPS-induced lung inflam-
mation.
6
Inhibiting the function of cyclophilins reduced the
influx of neutrophils by 40%–50% in this model.
6
Given the
many other neutrophil-recruiting chemokines known to be
present following LPS challenge,
12
we investigated in the
current studies whether cyclophilins might have the capacity
to synergize with MIP-2 and/or KC, the major neutrophil-
attracting chemokines in this disease.
In initial in vitro chemotaxis studies we observed a potent
functional interaction between CypA and MIP-2, but not
7.5%
4.7%
10.5%
Time (seconds)
CypA
MIP-2
CypA
+
MIP-2
1.0442
6.5312
12.656
18.742
24.858
30.904
36.99
43.136
49.242
55.308
Figure 4 
 
6
       °   


  

2+




2+
==+=

Abbreviations:
Line Group MFI
CypA + MIP-2 779
MIP-2 574
CypA 667
Medium
0
Phalloidin
Relative cell number
10
1
10
2
10
3
10
4
10
4
615
Line Group MFI
CypA + KC 394
KC 366
CypA 421
Medium
0
Phalloidin
Relative cell number
% increase in phalloidin MFI
10
1
10
2
10
3
10
4
CypA MIP-2
*
*
KC CypA
+
KC
CypA
+
MIP-2
10
4
−10
0
10
20
30
40
50
352
A
B
C
Figure 5         
           
            
 °  A            
+B
+
       
MFI were calculated and are shown for each group in panel legends. C) Bar graph
showing average increases in phalloidin expression relative to medium that were
calculated using 2 independent sets of data.
Abbreviations:

submit your manuscript | www.dovepress.com
Dovepress
Dovepress
101

cell type.
34
Our data fit this criterion in that the synergistic
effect was seen only at very specific dose combinations.
To provide support that the apparent synergistic interaction
between CypA and MIP-2 was also associated with altered
signaling events, several biochemical parameters were
measured. Combinations of CypA and MIP-2 did indeed
lead to an augmented internalization of CXCR2, increased
and more prolonged calcium mobilization, and enhanced
actin polymerization in neutrophils, although these changes
were for the most part additive rather than synergistic.
Similar discrepancies in functional outcome versus signal-
ing profiles have been reported during chemokine synergy
by other laboratories.
35,36
One possibility is that the specific
parameters measured in the current studies are not directly
associated with the signaling events required to mediate
functional synergy between chemokines. However, a more
likely explanation is that synergistic outcomes are due to the
integration of multiple signaling events. Thus, while changes
in one signaling parameter provide evidence for enhanced
receptor-mediated events, these events may represent only
one arm of an integrated signaling pathway.
Based on our current findings, we propose the following
mechanism for the augmented activation of neutrophils by
combinations of CypA and MIP-2. When the 2 chemoattrac-
tants are present together, CypA may promote greater and/
or more efficient binding of MIP-2 to its receptor CXCR2,
resulting in increased CXCR2 internalization (Figure 3).
The internalized receptor-chemokine (CXCR2-MIP-2)
complexes may then contribute to increased signaling by
promoting receptor recycling. Increases in downstream sig-
naling events are evidenced by an augmentation in calcium
flux (Figure 4). The ensuing increase in actin polymerization
(Figure 5) results in a more efficient migration by individual
cells, or enables a greater total number of cells to migrate, due
to previously sub-optimal thresholds of activation becom-
ing optimal. This is supported by in vivo observations that
neutrophil numbers are still high as late as 24 hours after LPS
delivery, yet inhibiting cyclophilins at this time point (when
cyclophilins are at their peak level) reduces neutrophilia
by only 40% to 50%.
6
Co-inhibiting CypA and MIP-2 (or
any additional chemokines found to cooperate with CypA)
provides a potential approach whereby late neutrophil recruit-
ment might be further reduced, resulting in a decrease in the
amplitude of the response and the collateral tissue damage
associated with prolonged neutrophilia. Indeed, such an out-
come was reported when the synergizing chemokines, IL-8,
and GM-CSF, were co-inhibited,
28
providing preliminary
support for this type of therapeutic intervention.
with KC, that resulted in augmented neutrophil migration.
Although several different doses of KC were tested and failed
to cooperate with CypA in the combination assays (data not
shown), we cannot rule out the possibility that KC and CypA
might be able to function in either an additive or synergistic
partnership at doses not tested here. Nevertheless, the current
findings suggest that partnerships between chemoattractants
are likely selective. Recent studies suggest that the family
to which a chemokine belongs is a critical parameter in
determining whether cooperative interactions will occur.
Specifically, these studies showed that chemokines belonging
to the same family (for example CXC or CC) are less likely
to interact due to their competing for related receptors and/
or using similar signaling pathways.
20
Both MIP-2 and KC
signal through the same receptor, CXCR2, suggesting that
the interaction with CypA may occur at a step preceding
receptor binding. A possible mechanism is that interactions
between MIP-2 and CypA enhance MIP-2 binding and/or
signaling through CXCR2, possibly via the peptidyl-prolyl
isomerization activity of CypA.
The principal binding and signaling receptor for CypA
is CD147, a type I integral membrane protein carrying 2
immunoglobulin-like domains.
30
Since CD147 bears no
structural homology with any of the 4 major families of
chemokine receptors (CR, CCR, CXCR, and CX3CR), extra-
cellular cyclophilins may be less restricted in their capacity
to interact and synergize with other chemokines. However,
it should be noted that the interaction between CypA/CypB
and CD147 has also been shown to require the presence of
cell surface heparans for optimal induction of cyclophilin
binding and signaling.
15,31
Recent studies have reported that
syndecan-1 is the principal heparan co-receptor for CypB,
which coordinates with CD147 to activate signaling pathways
and induce T cell adhesion and migration.
32
Interestingly,
KC has also been shown to bind syndecan-1.
33
The fact that
syndecan-1 is implicated in both cyclophilin and KC signal-
ing pathways could provide an alternative explanation for
why we did not observe any cooperation between these 2
chemoattractants in the current studies: CypA and KC may
compete for syndecan-1 binding, thus reducing their ability
to signal through the corresponding receptor and preventing
their capacity to interact.
Of particular interest was the observation that combina-
tions of CypA and MIP-2 mediated increases in neutrophil
migration that were greater than additive, suggesting a syn-
ergistic interaction. The phenomenon of synergy between
chemoattractants in vitro is typically observed only within
narrow ranges of concentrations that greatly depend on the

submit your manuscript | www.dovepress.com
Dovepress
Dovepress
102
Heine et al
Abbreviations
Ab, antibody; BAL, bronchoalveolar lavage; CsA,
cyclosporine A; CypA, cyclophilin A; dATP, deoxyadenosine
triphosphate; ELISA, enzyme-linked immunosorbent assay;
FITC, fluorescein isothiocyanate; FLIPR, fluorescence
imaging plate reader; fMLP, formyl-methionyl-leucyl-
phenylalanine; GM-CSF, granulocyte-macrophage colony-
stimulating factor; GCP, granulocyte chemoattractant protein;
GPCR, G protein-coupled receptor; HRP, horseradish
peroxidase; Ig, immunoglobulin; IP, interferon gamma-
induced protein; IL, interleukin; KC, keratinocyte chemoat-
tractant; LIX, lipopolysaccharide-induced CXC chemokine;
LPS, lipopolysaccharide; mAB, monoclonal antibody;
MCP, monocyte chemotactic protein; MDC, macrophage-
derived chemokine; MFI, mean fluorescence intensity;
MIP, macrophage inflammatory protein; NAP, neutrophil
activating protein; PBMC, peripheral blood mononuclear
cell; PBS, phosphate buffered saline; PE, phycoerythrin;
RANTES, regulated on activation, normally T cell expressed
and secreted; RBC, red blood cell; SDS-PAGE, sodium
dodecyl sulfate polyacrylamide gel electrophoresis; SE,
standard error.
Acknowledgment
These studies were supported by NIH grant R01AI067254.
Disclosure
The authors report no conflicts of interest in this work.
References
1. Sherry B, Yarlett N, Strupp A, Cerami A. Identification of cyclophilin
as a proinflammatory secretory product of lipopolysaccharide-activated
macrophages. Proc Natl Acad Sci U S A. 1992;89(8):3511–3515.
2. Tegeder I, Schumacher A, John S, et al. Elevated serum cyclophilin
levels in patients with severe sepsis. J Clin Immunol. 1997;17(5):
380–386.
3. Jin ZG, Melaragno MG, Liao DF, et al. Cyclophilin A is a secreted growth
factor induced by oxidative stress. Circ Res. 2000;87(9):789–796.
4. Billich A, Winkler G, Aschauer H, Rot A, Peichl P. Presence of cyclo-
philin A in synovial fluids of patients with rheumatoid arthritis. J Exp
Med. 1997;185(5):975–980.
5. Jin ZG, Lungu AO, Xie L, Wang M, Wong C, Berk BC. Cyclophilin A is
a proinflammatory cytokine that activates endothelial cells. Arterioscler
Thromb Vasc Biol. 2004;24(7):1186–1191.
6. Arora K, Gwinn WM, Bower MA, et al. Extracellular cyclophilins
contribute to the regulation of inflammatory responses. J Immunol.
2005;175(1):517–522.
7. Gwinn WM, Damsker JM, Falahati R, et al. Novel approach to inhibit
asthma-mediated lung inflammation using anti-CD147 intervention.
J Immunol. 2006;177(7):4870–4879.
8. Damsker JM, Okwumabua I, Pushkarsky T, Arora K, Bukrinsky MI,
Constant SL. Targeting the chemotactic function of CD147 reduces
collagen-induced arthritis. Immunology. 2009;126(1):55–62.
9. Bukrinsky MI. Cyclophilins: unexpected messengers in intercellular
communications. Trends Immunol. 2002;23(7):323–325.
10. Greenberger MJ, Strieter RM, Kunkel SL, et al. Neutralization of
macrophage inflammatory protein-2 attenuates neutrophil recruitment
and bacterial clearance in murine Klebsiella pneumonia. Journal of
Infectious Diseases. 1996;173(1):159–165.
11. McColl SR, Clark-Lewis I. Inhibition of murine neutrophil recruit-
ment in vivo by CXC chemokine receptor antagonists. J Immunol.
1999;163(5):2829–2835.
12. Jeyaseelan S, Chu HW, Young SK, Worthen GS. Transcriptional pro-
filing of lipopolysaccharide-induced acute lung injury. Infect Immun.
2004;72(12):7247–7256.
13. Maus U, von Grote K, Kuziel WA, et al. The role of CC chemokine
receptor 2 in alveolar monocyte and neutrophil immigration in intact
mice. Am J Respir Crit Care Med. 2002;166(3):268–273.
14. Gouwy M, Struyf S, Proost P, Van Damme J. Synergy in cytokine and
chemokine networks amplifies the inflammatory response. Cytokine
Growth Factor Rev. 2005;16(6):561–580.
15. Saphire AC, Bobardt MD, Gallay PA. Host cyclophilin A mediates
HIV-1 attachment to target cells via heparans. Embo J. 1999;18(23):
6771–6785.
16. Yurchenko V, Zybarth G, O’Connor M, et al. Active site residues of
cyclophilin A are crucial for its signaling activity via CD147. J Biol
Chem. 2002;277(25):22959–22965.
17. Gessner PK. Isobolographic analysis of interactions: an update on
applications and utility. Toxicology. 1995;105(2–3):161–179.
18. Mellado M, Rodriguez-Frade JM, Vila-Coro AJ, et al. Chemokine recep-
tor homo- or heterodimerization activates distinct signaling pathways.
Embo J. 2001;20(10):2497–2507.
19. Yurchenko V, O’Connor M, Dai WW, et al. CD147 is a signaling recep-
tor for cyclophilin B. Biochem Biophys Res Commun. 2001;288(4):
786–788.
20. Gouwy M, Struyf S, Noppen S, et al. Synergy between coproduced
CC and CXC chemokines in monocyte chemotaxis through receptor-
mediated events. Mol Pharmacol. 2008;74(2):485–495.
21. Kuscher K, Danelon G, Paoletti S, et al. Synergy-inducing chemok-
ines enhance CCR2 ligand activities on monocytes. Eur J Immunol.
2009;39(4):1118–1128.
22. Sebastiani S, Danelon G, Gerber B, Uguccioni M. CCL22-induced
responses are powerfully enhanced by synergy inducing chemokines via
CCR4: evidence for the involvement of first beta-strand of chemokine.
Eur J Immunol. 2005;35(3):746–756.
23. Segel GB, Halterman MW, Lichtman MA. The paradox of the neutro-
phil’s role in tissue injury. J Leukoc Biol. 2011;89(3):359–372.
24. Lonkar P, Dedon PC. Reactive species and DNA damage in chronic
inflammation: reconciling chemical mechanisms and biological fates.
Int J Cancer. 2011;128(9):1999–2009.
25. Struyf S, Proost P, Lenaerts JP, Stoops G, Wuyts A, Van Damme J.
Identification of a blood-derived chemoattractant for neutrophils and
lymphocytes as a novel CC chemokine, Regakine-1. Blood. 2001;
97(8):2197–2204.
26. Gouwy M, Struyf S, Mahieu F, Put W, Proost P, Van Damme J. The
unique property of the CC chemokine regakine-1 to synergize with other
plasma-derived inflammatory mediators in neutrophil chemotaxis does
not reside in its NH2-terminal structure. Mol Pharmacol. 2002;62(1):
173–180.
27. Struyf S, Gouwy M, Dillen C, Proost P, Opdenakker G, Van Damme J.
Chemokines synergize in the recruitment of circulating neutrophils into
inflamed tissue. Eur J Immunol. 2005;35(5):1583–1591.
28. Shen L, Fahey JV, Hussey SB, Asin SN, Wira CR, Fanger MW.
Synergy between IL-8 and GM-CSF in reproductive tract epithelial cell
secretions promotes enhanced neutrophil chemotaxis. Cell Immunol.
2004;230(1):23–32.
29. Stanford MM, Issekutz TB. The relative activity of CXCR3 and CCR5
ligands in T lymphocyte migration: concordant and disparate activities
in vitro and in vivo. J Leukoc Biol. 2003;74(5):791–799.
30. Yurchenko V, Constant S, Bukrinsky M. Dealing with the family: CD147
interactions with cyclophilins. Immunology. 2006;117(3):301–309.

submit your manuscript | www.dovepress.com
Dovepress
Dovepress
103

31. Denys A, Allain F, Carpentier M, Spik G. Involvement of two classes
of binding sites in the interactions of cyclophilin B with peripheral
blood T-lymphocytes. Biochem J. 1998;336(Pt 3):689–697.
32. Pakula R, Melchior A, Denys A, Vanpouille C, Mazurier J, Allain F.
Syndecan-1/CD147 association is essential for cyclophilin B-induced
activation of p44/42 mitogen-activated protein kinases and promotion
of cell adhesion and chemotaxis. Glycobiology. 2007;17(5):492–503.
33. Li Q, Park PW, Wilson CL, Parks WC. Matrilysin shedding of
syndecan-1 regulates chemokine mobilization and transepithelial efflux
of neutrophils in acute lung injury. Cell. 2002;111(5):635–646.
34. Gouwy M, Struyf S, Verbeke H, et al. CC chemokine ligand-2 syner-
gizes with the nonchemokine G protein-coupled receptor ligand fMLP
in monocyte chemotaxis, and it cooperates with the TLR ligand LPS
via induction of CXCL8. J Leukoc Biol. 2009;86(3):671–680.
35. El-Asmar L, Springael JY, Ballet S, Andrieu EU, Vassart G,
Parmentier M. Evidence for negative binding cooperativity within
CCR5-CCR2b heterodimers. Mol Pharmacol. 2005;67(2):460–469.
36. Gouwy M, Struyf S, Catusse J, Proost P, Van Damme J. Synergy
between proinflammatory ligands of G protein-coupled receptors in neu-
trophil activation and migration. J Leukoc Biol. 2004;76(1):185–194.
Journal of Inflammation Research
Publish your work in this journal
Submit your manuscript here: l
The Journal of Inflammation Research is an international, peer-reviewed
open-access journal that welcomes laboratory and clinical findings on the
molecular basis, cell biology and pharmacology of inflammation including
original research, reviews, symposium reports, hypothesis formation and
commentaries on: acute/chronic inflammation; mediators of inflamma-
tion; cellular processes; molecular mechanisms; pharmacology and novel
anti-inflammatory drugs; clinical conditions involving inflammation.
The manuscript management system is completely online and includes a
very quick and fair peer-review system. Visit http://www.dovepress.com/
testimonials.php to read real quotes from published authors.

submit your manuscript | www.dovepress.com
Dovepress
Dovepress
Dovepress
104
Heine et al
4
1
2
3
3
2
1
1
2
3
4
Chemotactic indexChemotactic indexChemotactic index
A
B
C
fMLP 10 20 50 100
fMLP 10 20 100 200
fMLP 5102050
MIP-2 (ng/mL)
KC (ng/mL)
CypA (ng/mL)
KC
MIP-2
CypA
Figure S1 

incubated in the presence of a single dose of fMLP (positive control) and increasing

by dividing the number of migrated cells in test wells by the number of cells that
migrated to medium alone. A) Mean ±     
B) Mean ±
C) Mean ±

= 6 wells for each group.
Abbreviations:
Supplementary gure
    • "Macrophage inflammatory protein 2 (MIP-2), the mouse analogue of the human pro-inflammatory cytokine IL-8, is a potent neutrophil chemoattractant . Together with CypA, MIP-2 was demonstrated to have a synergistic effect on neutrophil migration [27]. In humans, CypA induces the secretion of the pro-inflammatory cytokine IL-8. "
    [Show abstract] [Hide abstract] ABSTRACT: Introduction and aim: The role of B-lymphocytes in chemical-induced asthma is largely unknown. Recent work demonstrated that transferring B lymphocytes from toluene diisocyanate (TDI)-sensitized mice into naïve mice, B cell KO mice and SCID mice, triggered an asthma-like response in these mice after a subsequent TDI-challenge. We applied two-dimensional difference gel electrophoresis (2D-DIGE) to describe the "sensitized signature" of B lymphocytes comparing TDI-sensitized mice with control mice. Results: Sixteen proteins were identified that were significantly up- or down-regulated in B lymphocytes of sensitized mice. Particularly differences in the expression of cyclophilin A, cofilin 1 and zinc finger containing CCHC domain protein 11 could be correlated to the function of B lymphocytes as initiators of T lymphocyte independent asthma-like responses. Conclusion: This study revealed important alterations in the proteome of sensitized B cells in a mouse model of chemical-induced asthma, which will have an important impact on the B cell function.
    Full-text · Article · Sep 2015
    • "Blocking CyP-CD147 interactions by either anti-CD147 antibody or by CsA alone reduced tissue neutrophils levels by almost half [98]. Combining CyPA with the classical chemokine macrophage inflammatory protein (MIP2) enhances neutrophil migration which synergistically increases its receptor internalization, calcium mobilization and actin polymerization [99]. Neutrophil-mediated phagocytosis of invading pathogens is another important aspect of neutrophil-mediated host immune surveillance, and this function is enhanced in the presence of granulocyte macrophage colony-stimulating factor (GM-CSF) and interleukin-8 (IL-8). "
    [Show abstract] [Hide abstract] ABSTRACT: Peptidyl-prolyl isomerase (PPIase) catalyzes the interconversion of a specific Pro-imide bond between the cis and trans conformations. Two families of PPIases, cyclophilins and FKBPs, have been extensively studied because of their high affinity for immunosuppressive drugs in particular cyclosporine A and FK506. Despite apparent differences, these protein families share conserved amino acid sequences in their catalytic domains and impose similar enzymatic functions to their substrates. PPIases have been implicated in multiple aspects of cell cycle regulation and cellular processes related to a number of human pathologies, including cancer. More recent studies provide evidence for participation of PPIases in regulation of immune cell functions. In this review, we focus on the role of cyclophilins and FKBPs in the regulation of innate and adaptive immunity functions. PPIase-mediated isomerization of proteins represents a unique signaling mechanism that regulates normal immune functions and contributes to the development of immunopathologies. PPIases may therefore serve as useful diagnostic tools and potential therapeutic targets. Copyright © 2014. Published by Elsevier B.V.
    Full-text · Article · Nov 2014
    • "The content of some cytokines varied greatly; for example, the expressions of MIP-2, IL-6, and GRO in UC-MSC-CM were significantly higher than those in ASC-CM, while the expressions of CD27 and neuregulin in ASC-CM were significantly higher than those in UC-MSC- CM. MIP-2 is the main chemotactic cytokine of neutrophil, and it can specifically promote neutrophil migrating to the inflammatory tissue, to get rid of pathogens and participate in the body's defense reaction [35]; MMP-1 is involved in mediation of a wide range of physiological and pathological processes in the body, such as the formation of embryo, tissue remodeling, wound healing, inflammation, and apoptosis [36]. IL-6 can promote the proliferation of a variety of cells, and this might be one of the reasons for UC-MSCs proliferating faster than ASCs [37]. "
    [Show abstract] [Hide abstract] ABSTRACT: Both human adipose tissue-derived mesenchymal stem cells (ASCs) and umbilical cord-derived mesenchymal stem cells (UC-MSCs) have been explored as attractive mesenchymal stem cells (MSCs) sources, but very few parallel comparative studies of these two cell types have been made. We designed a side-by-side comparative study by isolating MSCs from the adipose tissue and umbilical cords from mothers delivering full-term babies and thus compared the various biological aspects of ASCs and UC-MSCs derived from the same individual, in one study. Both types of cells expressed cell surface markers characteristic of MSCs. ASCs and UC-MSCs both could be efficiently induced into adipocytes, osteoblasts, and neuronal phenotypes. While there were no significant differences in their osteogenic differentiation, the adipogenesis of ASCs was more prominent and efficient than UC-MSCs. In the meanwhile, ASCs responded better to neuronal induction methods, exhibiting the higher differentiation rate in a relatively shorter time. In addition, UC-MSCs exhibited a more prominent secretion profile of cytokines than ASCs. These results indicate that although ASCs and UC-MSCs share considerable similarities in their immunological phenotype and pluripotentiality, certain biological differences do exist, which might have different implications for future cell-based therapy.
    Full-text · Article · Jul 2013
Show more

Supplementary resources