IL-1α and IL-1β Recruit Different Myeloid
Cells and Promote Different Stages of Sterile
Inflammation
This information is current as
of June 15, 2017.
Peleg Rider, Yaron Carmi, Ofer Guttman, Alex Braiman,
Idan Cohen, Elena Voronov, Malka R. White, Charles A.
Dinarello and Ron N. Apte
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Copyright © 2011 by The American Association of
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Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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J Immunol 2011; 187:4835-4843; Prepublished online 19
September 2011;
doi: 10.4049/jimmunol.1102048
http://www.jimmunol.org/content/187/9/4835
The Journal of Immunology
IL-1a and IL-1b Recruit Different Myeloid Cells and
Promote Different Stages of Sterile Inflammation
Peleg Rider,*,1 Yaron Carmi,*,1 Ofer Guttman,* Alex Braiman,* Idan Cohen,*
Elena Voronov,* Malka R. White,* Charles A. Dinarello,† and Ron N. Apte*
T
he immune system evolved to protect the host from invading pathogens and to restore tissue homeostasis after
injury or damage. The immune response against pathogens
occurs via tissue-associated phagocytes, which discriminate selffrom nonself through pattern-recognition receptors. Such recognition is subsequently followed by maturation to macrophages,
which in turn initiate and direct the inflammatory/immune response
against nonself molecules (1). In contrast, the response to tissue
injury requires recognition of alarm signals in a sterile environment by cells of the innate immune system. As a result, myeloid
cells are recruited into damaged tissue. Initially, neutrophils are
recruited (2), followed by phagocytes of the mononuclear lineage,
which differentiate and redefine their transcriptome to express
genes involved in tissue repair (3). The identity of cell components
*The Shraga Segal Department of Microbiology and Immunology and The Cancer
Research Center, Faculty of Health Sciences, Ben-Gurion University of the Negev,
Beer Sheva 84105, Israel; and †Department of Medicine, University of Colorado
Health Sciences Center, Denver, CO 80262
1
P.R. and Y.C. contributed equally to this work.
Received for publication July 15, 2011. Accepted for publication August 22, 2011.
This work was supported by the Israel Ministry of Science jointly with the Deutsches
Krebsforschungscentrum, the Israel Science Foundation (founded by the Israel Academy of Sciences and Humanities), and the Deutsch-Israelische Projekttkooperation
(to R.N.A.). R.N.A. is an incumbent of the Irving Isaac Sklar Chair in Endocrinology
and Cancer. E.V. was supported by the Israel Cancer Association, the Israel Ministry
of Health Chief Scientist’s Office, the Concern Foundation, the Deutsch-Israelische
Projekttkooperation, FP7 INFLA-CARE program: Cancer and Inflammation, and the
Israel Science Foundation (the last three in cooperation with R.N.A.).
Address correspondence and reprint requests to Dr. Ron N. Apte, Ben-Gurion University of the Negev, Faculty of Health Sciences, P.O. Box 653, Beer Sheva 84105,
Israel. E-mail address: [email protected]
The online version of this article contains supplemental material.
Abbreviations used in this article: HMGB1, high-mobility group box-1; IL-1Ra, IL1R antagonist; IL-1RI, type I IL-1R; KO, knockout; PI, propidium iodide; WT, wildtype.
Copyright Ó 2011 by The American Association of Immunologists, Inc. 0022-1767/11/$16.00
www.jimmunol.org/cgi/doi/10.4049/jimmunol.1102048
that are released exclusively during necrosis and how the subsequent inflammatory response is orchestrated to restore tissue
homeostasis are under current extensive investigations.
Most cells are vulnerable to hypoxic conditions due to the loss of
mitochondrial respiration and acidosis (4). Because tissue injury is
usually associated with insufficient blood supply and ischemia,
the microenvironment of various pathologies involves anaerobic
metabolites and products of necrotic cells (5, 6). In turn, necrotizing cells lose their membrane integrity, resulting in the release
of intracellular components that stimulate the immune response
through damage-associated molecular pattern receptors (7). Over
the past decade, a number of endogenous molecules, now known as
alarmins, have been identified by their ability to induce leukocyte
recruitment and activation and also to cause maturation of APCs
(8). Most alarmins are constitutively expressed under homeostatic
conditions and released upon necrosis (9–14), but not apoptosis
(13, 15, 16). Surprisingly, endogenous alarmins can interact with
TLR (heat shock proteins, high mobility group box 1 [HMGB1]) or
type I IL-1R (IL-1RI), which are also key receptors in recognizing
and directing the immune response against microbial pathogens.
In a key study by Chen et al. (9), it was demonstrated that products
of necrotic cells stimulate the immune system in an IL-1RI–
dependent manner rather than through TLR signaling.
The IL-1 molecules comprise a major proinflammatory family of
cytokines, which act mainly through the induction of a complex
network of proinflammatory cytokines/mediators and via expression of integrins on leukocytes and endothelial cells (17–20). Of the
11 members of the IL-1 family of ligands, IL-1a and IL-1b are the
two major agonistic molecules, whereas the IL-1R antagonist (IL1Ra) is a physiological inhibitor of preformed IL-1. IL-1a, IL-1b,
and the IL-1Ra bind to IL-1RI signaling receptor. Although both
IL-1a and IL-1b trigger inflammation in a pathway initiated
through Myd88 activation and culminated in NF-kB–induced
transcription of inflammatory genes, several findings suggest that
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The immune system has evolved to protect the host from invading pathogens and to maintain tissue homeostasis. Although the
inflammatory process involving pathogens is well documented, the intrinsic compounds that initiate sterile inflammation and
how its progression is mediated are still not clear. Because tissue injury is usually associated with ischemia and the accompanied
hypoxia, the microenvironment of various pathologies involves anaerobic metabolites and products of necrotic cells. In the current
study, we assessed in a comparative manner the role of IL-1a and IL-1b in the initiation and propagation of sterile inflammation
induced by products of hypoxic cells. We found that following hypoxia, the precursor form of IL-1a, and not IL-1b, is upregulated
and subsequently released from dying cells. Using an inflammation-monitoring system consisting of Matrigel mixed with supernatants of hypoxic cells, we noted accumulation of IL-1a in the initial phase, which correlated with the infiltration of neutrophils,
and the expression of IL-1b correlated with later migration of macrophages. In addition, we were able to show that IL-1 molecules
from cells transfected with either precursor IL-1a or mature IL-1b can recruit neutrophils or macrophages, respectively. Taken
together, these data suggest that IL-1a, released from dying cells, initiates sterile inflammation by inducing recruitment of
neutrophils, whereas IL-1b promotes the recruitment and retention of macrophages. Overall, our data provide new insight into
the biology of IL-1 molecules as well as on the regulation of sterile inflammation. The Journal of Immunology, 2011, 187: 4835–
4843.
4836
Materials and Methods
Mice
Wild-type (WT) C57BL/6 mice were obtained from Harlan Laboratories.
Other strains used were: IL-1a and IL-1b knockout (KO) mice (27),
caspase-1 KO mice (28), IL-18BP transgenic mice (29), and IL-1RI KO
mice purchased from The Jackson Laboratory (Bar Harbor, ME). The KO
mice are homogenous for the relevant mutations and were backcrossed
with WT C57BL/6 mice as described to maintain identical genetic background (27, 28). Animal studies were approved by the Animal Care
Committee of Ben-Gurion University of the Negev (Beer Sheva, Israel).
Male 6–8-wk-old mice were used in all experiments.
Cell culture
BD7 cells were cultured in a sealed anaerobic workstation (Concept 400;
Ruskinn Technology/Jouan) providing hypoxic conditions (O2 ,0.3%, 5%
CO2, 95% N2, and 37˚C). After 24 h incubation, the cells were collected
and analyzed by Annexin V/propidium iodide (PI) staining (Bender
MedSystems). The cell medium was collected and analyzed for IL-1a and
IL-1b by ELISA (R&D Systems and BD Pharmingen).
HEK-T293 cells were transfected using the calcium phosphate technique
in which transfection solution was made with 400 ml CaCl2 (250 mM),
which was mixed with 8 mg plasmid DNA, and then was dropped gently
into 400 ml HEPES-buffered saline (32 concentrated) during gentle agitation to produce aggregates. Next, the solution was added to the cell
culture media. The plasmids added to the transfection solution are the IL1a and IL-1b pEGFP-N1 constructs described below. After 24 h, cells
were washed with PBS, lysed by four cycles of freeze-thawing, centrifuged, and supernatants were collected.
To obtain cells from different organs, mice were sacrificed, and heart,
kidney, liver, and lungs were removed, digested with collagenase II (heart)
or collagenase V at 37˚C for 1 h, with stirring. Cells were separated using
a 70-mm cell strainer (BD Pharmingen), and lysed by four cycles of freezethawing.
Constructs of IL-1–GFP
IL-1b cDNA was amplified by PCR using forward (59-CAAGCTTGCCACCATGCACTACAGG-39) and reverse (59-CGGTACCGCGGAAGACACG-39) primers cloned into the PGEM-T easy vector (Promega). Next,
inserts were sequenced and cloned into pEGFP-N1 (Clontech) using
HindIII and KpnI restriction sites. The IL-1a precursor was cloned with
GFP as described (26).
The Matrigel plug assay of inflammation
Hypoxic or normoxic cell medium (40 ml) or, alternatively, 5 mg HEKT293 transfected lysate or 20 mg total lysate from cells obtained from
mouse tissues, was mixed with liquid Matrigel (500 ml; BD Biosciences)
and injected subcutaneously into IL-1a KO, IL-1b KO, or WT mice. For
IL-1 neutralization, 1 mg/mouse IL-1Ra (Amgen) or neutralizing Abs
(1 mg/mouse), anti–IL-1a (AF-400-NA; R&D Systems), anti–IL-1b (AF401-NA; R&D Systems), or goat IgG (R&D Systems) were mixed with the
Matrigel (10 mg/mouse; BD Biosciences).
To isolate infiltrating cells, Matrigel plugs (BD Biosciences) were removed from mice and incubated with an enzyme mixture containing 1 g/100
ml collagenase type IV, 20,000 units/100 ml DNase type IV, and 1 mg/ml
hyaluronidase type V (Sigma-Aldrich, Rehovot, Israel) in 2 ml HBSS (Life
Technologies), at 37˚C for 1 h, as described (18). Cells were counted using
an automated cell counter (Digital Bio).
Detection of IL-1a and IL-1b
Lysates of BD7 cells were separated on PAGE, transferred to nitrocellulose
membranes (Whatman), and blotted with goat anti–C-terminal IL-1a Abs
(AF-400-NA; R&D Systems) or with polyclonal rabbit anti–IL-1a
N-terminal peptide Abs raised against the 21-aa sequence CYSENEDYSSAIDHLSLNQKS. To measure the total protein load, mouse anti–bactin (691001; MP Biomedicals) was used. Additionally, transfected HEKT293 cell lysates were blotted with hamster anti–IL-1b (MAB4012; R&D
Systems) and mouse anti-GFP (MMS-118P; Covance).
Total RNA was extracted from the cells (RNeasy; Qiagen, Valencia, CA)
and quantified using a NanoDrop (ND-1000 spectrophotometer; NanoDrop
Technologies). cDNA Reverse-Transcription was performed with 1 mg total
RNA as a template using random hexamers and MuLV reverse transcriptase (Applied Biosystems, Foster City, CA). Subsequently, IL-1a expression was monitored using the Real Time PCR ABI Prism 7500 sequence
detection system (Applied Biosystems). Primers used for real-time PCR
were b-actin: forward, 59-GGTCTCAAACATGATCTGGG-39 and reverse,
59-GGGTCAGAAGGATTCCTATG-39; and IL-1a: forward, 59-CCGAGTTTCATTGCCTCTTT-39 and reverse, 59-ACTGTGGGAGTGGAGTGCTT-39.
Confocal microscopy
BD7 cells and HEK-T293–transfected cells were plated and observed in
Fluorodish plates (World Precision Instruments). BD7 cells were fixed with
4% paraformaldehyde, permeabilized with 0.5% Triton X-100, and stained
with 1:200 diluted goat anti–IL-1a Ab (AF-400-NA; R&D Systems) and
secondary donkey anti-goat Cy3 (Jackson ImmunoResearch Laboratories).
For frozen sections, immunofluorescent stained Matrigel plugs (BD
Biosciences) were fixed in 4% paraformaldehyde for 6 h and equilibrated in
a 20% sucrose solution for 24 h, embedded in frozen tissue matrix (TissueTek OCT, Torrance, CA), and frozen at -280˚C. The 12-mm-thick sections
were blocked with 20% nonimmune goat serum and stained with 1:50
diluted goat anti–IL-1b (AF-401-NA; R&D Systems), goat anti–IL-1a
(AF-400-NA; R&D Systems), or 1:100 diluted rat anti-CD11b (M1/70;
eBioscience) Abs. Specimens were subsequently stained with secondary
Abs conjugated with either Cy2 or Cy3 (Jackson ImmunoResearch Laboratories). Nuclei were counterstained with DAPI.
Cells were examined using a 405-nm excitation laser line for either DAPI
in fixed cells or Hoechst 33342 (Fluka) in live cells, a 488-nm laser line for
GFP in live HEK-T293 cells or Cy2 in frozen sections, and a 559-nm laser
line for Cy3 in fixed BD7 cells. Microscopy was performed with an
Olympus Fluoview FV1000 confocal microscope (Olympus).
Flow cytometry
Single-cell suspensions obtained from Matrigel plugs (BD Biosciences)
were analyzed using flow cytometry (FACSCanto; BD Biosciences).
Datasets were analyzed using FlowJo software (Tree Star). mAbs conjugated to FITC, PE, PE-Cy7, PE-Cy5.5, allophycocyanin, allophycocyaninCy7, or Pacific Blue specific for the following Ags were used: CD11b
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both IL-1 agonists may have different activities. IL-1b is not
expressed under normal homeostatic conditions by bone marrowderived myeloid cells and is active only upon cleavage of its
precursor by caspase-1 on the inflammasome, which also leads to
its secretion (20, 21). Clinical and experimental observations have
correlated IL-1b to pathologies in which inflammatory manifestations are observed. IL-1a, unlike IL-1b, can be found constitutively inside cells under normal homeostasis, and it is active in
its precursor as well as calpain-processed mature form (21). IL-1a
has a functional nuclear localization sequence and is active in the
intracellular compartment, especially in the nucleus, as a transcription regulator, whereas it affects inflammation and immunity
outside the cell. Thus, IL-1a belongs to a group of dual-function
cytokines, together with IL-33, HMGB1, and IL-37 (12, 22–25).
Nevertheless, the possibility of differences in function of IL-1a
and IL-1b during sterile inflammation is still hypothetical. Previous reports suggest that IL-1a serves as an alarm signal for
initiating inflammation in response to tissue injury (9, 10). IL-1a
is present in the skin, wherein injuries lead to a hypoxic state,
resulting in massive cell necrosis (5–7). Indeed, we recently
demonstrated that IL-1a is released from necrotic cells, whereas
apoptotic cells retained IL-1a within the nucleus as a mechanism
to preserve immune tolerance and prevent inflammation (26).
In this paper, we have studied the differential effects of IL-1a
and IL-1b on the initiation and duration of inflammation induced
by products of necrotic cells. By using the Matrigel plug (BD
Biosciences) model of sterile inflammation, we have demonstrated
that following hypoxia-mediated IL-1a release, IL-1a and IL-1b
are further expressed at different phases of the inflammatory response by neutrophils and macrophages, respectively. Furthermore,
different subsets of myeloid cell were found to be recruited following stimulation by cell-derived IL-1a or IL-1b, suggesting that
each of these IL-1R agonists induces a distinct and unique response in inflammation. Better understanding these functions may
enable future approaches to more accurately attenuate inflammation in different pathologies.
STERILE INFLAMMATION IS IL-1 DEPENDENT
The Journal of Immunology
4837
(M1/70), Gr-1 (RB6-8C5), Ly6C (AL-21), and F4/80 (BM8) (eBioscience,
San Diego, CA). Anti-CXCR4 (247506) Abs were purchased from R&D
Systems. Anti-Ly6G (IA8) Abs were purchased from BioLegend (San
Diego, CA). Cells were suspended in FACS buffer consisting of PBS with
3% FCS and 0.01% NaN3. Dead cells were excluded by use of the Live/
Dead Fixable Aqua Dead Cell Stain kit (Invitrogen). Intracellular cytokine
staining in permeabilized cells was performed, using anti–IL-1b (11-7113)
and anti–IL-1a Abs (12-7011), which were purchased from eBioscience.
Necrosis was evaluated by Annexin V/PI staining (Bender MedSystems).
Statistical analyses
Each experiment was performed three times. In Matrigel plug assays (BD
Biosciences), each experimental group consisted of at least three mice.
Significance of results was determined using the nonparametric one-way
ANOVA, when multiple groups are analyzed, or nonparametric Student t
test.
Results
The precursor form of IL-1a is the active component that
recruits leukocytes when released from hypoxic cells
FIGURE 1. The precursor form of IL-1a is released from hypoxic cells
and recruits leukocytes into Matrigel plugs (BD Biosciences). A, Immunofluorescence staining of IL-1a (green) in BD7 keratinocytes cultured for
24 h under normoxic or hypoxic conditions (original magnification 3400).
B, Left panel, Relative quantification (RQ) of IL-1a mRNA in BD7 keratinocytes cultured for 24 h under normoxic or hypoxic conditions. Results
represent mean values 6 SEM of three biological repeats. *p , 0.05.
Center panel shows Western blot analysis of extracts from BD7 keratinocytes using anti–N-terminal IL-1a (top panel) or anti–C-terminal IL-1a Abs
(middle panel). Bottom panel shows b-actin as loading control. Mean 6
SEM of IL-1a relative increase (measured by band densitometry) from
three blots is represented in right panel. C, Flow cytometry analysis of
Annexin V/PI staining in 24 h normoxic or hypoxic BD7 keratinocytes. D,
Mean levels of IL-1a in supernatants of normoxic or hypoxic BD7 keratinocytes (left panel). Results represent average 6 SEM. **p , 0.01.
Supernatants of normoxic or hypoxic BD7 keratinocytes injected as such,
or with IL-1Ra, into WT mice (right panel). Graphs indicate the total number of infiltrating cells in Matrigel plugs (BD Biosciences) after 24 h. Data
shown are from a single experiment. Experiments were repeated three times,
and each experimental group consisted of three mice. Results represent the
average 6 SEM. **p , 0.01 compared with the hypoxia subgroup.
supernatants of normoxic BD7 cells. Significantly, a considerable
reduction in the number of infiltrating leukocytes into plugs was
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We recently reported that infiltration of leukocytes into Matrigel
(BD Biosciences) containing lysates of necrotic cells is IL-1a
dependent (26). Therefore, it was of interest to assess the role of
cell-associated IL-1a as an alarm cytokine under physiological
stress conditions. Previous reports (30), as well as our results
(Supplemental Fig. 3C), indicate that the precursor of IL-1a is
constitutively expressed in keratinocytes and other types of epithelial cells.
Because most tissue injuries are associated with decreased
perfusion and ischemia resulting in cellular hypoxic stress, we
cultured BD7 keratinocytes under normoxic and hypoxic conditions and examined their expression of IL-1a. As indicated by
immunofluorescent staining, both normoxic and hypoxic BD7
keratinocytes express intracellular IL-1a, with similar localization
of IL-1a in the cytoplasm and nucleus (Fig. 1A). In contrast, upon
apoptotic stress, the nuclear localization of IL-1a in BD7 keratinocytes is increased (Supplemental Fig. 1). Interestingly, yet
consistent with our previous reports (18, 31), IL-1a was upregulated under hypoxia, as indicated by mRNA transcripts and protein
levels (Fig. 1B). Most importantly, by using novel Abs against
both the N-terminal and C-terminal residues of IL-1a produced in
our laboratory, Western blot analysis of BD7 lysates clearly indicated that the dominant form of IL-1a is a 31-kDa precursor
rather than its processed 17-kDa form (Fig. 1B, middle panel).
Because alarmins are released from dying cells to signal danger,
we next examined the effects of continuous hypoxic conditions on
IL-1a release from BD7 keratinocytes. Initially, the level of necrosis was evaluated in BD7 keratinocytes exposed to hypoxia for
24 h. As indicated by PI staining, whereas only marginal levels
of necrosis were observed in normoxic BD7 cells, ∼95% of the
hypoxic BD7 cells were necrotic (Fig. 1C). Moreover, the levels of
IL-1a in supernatants from hypoxic BD7 cells were between four
and five times higher compared with supernatants from normoxic
BD7 cells (Fig. 1D, left panel). Significantly, IL-1b was not
detected under hypoxic or normoxic conditions, as detected by
ELISA (data not shown). We next assessed the capacity of
supernatants from hypoxic and normoxic BD7 cells to recruit
leukocytes in a Matrigel plug assay (BD Biosciences) of inflammation. Thus, Matrigel (BD Biosciences) was mixed with
supernatants from hypoxic or normoxic BD7 cells and injected s.c
into WT mice. Matrigel plugs (BD Biosciences) were removed
from mice 24 h following injection, enzymatically digested, and
the infiltrating cells were counted. As shown in Fig. 1D, the
number of leukocytes that infiltrate into Matrigel plugs (BD
Biosciences) containing supernatants of hypoxic BD7 was ∼4-fold
higher compared with Matrigel plugs (BD Biosciences) containing
4838
STERILE INFLAMMATION IS IL-1 DEPENDENT
observed upon addition of IL-1Ra to Matrigel (BD Biosciences)
containing hypoxic supernatants (Fig. 1D, right panel).
IL-1a and IL-1b affect different phases of sterile inflammation
IL-1a and IL-1b are expressed in different myeloid subsets
during inflammation
We next sought to assess the contribution of host-infiltrating
myeloid cells to IL-1a and IL-1b production in the inflamma-
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It was of interest to establish whether the initial recruitment of
leukocytes depends only on IL-1a derived from necrotizing cells
or whether IL-1 of host origin is also involved in it, as IL-1Ra
blocks the binding of both IL-1b and IL-1a, whether from necrotizing cells or host origin. For this purpose, Matrigel (BD Biosciences) containing supernatants of hypoxic BD7 keratinocytes
were injected as such or mixed with neutralizing Abs against IL1a or IL-1b, into WT, IL-1a, or IL-1b KO mice. As shown in Fig.
2A, Matrigel (BD Biosciences) mixed with hypoxic supernatants
induced a massive leukocyte infiltration 24 h following injection
to WT mice. In contrast, the number of leukocytes was markedly
reduced when anti–IL-1a Abs were added to the plugs (Fig. 2A).
Neutralizing IL-1b Abs reduce the infiltrating cell numbers but to
a much lesser extent (Fig. 2A). Interestingly, no significant reduction in the amount of the leukocyte infiltrate in Matrigel plugs
(BD Biosciences) was observed in IL-1a or IL-1b KO mice, indicating that IL-1a of necrotic cell origin, rather than IL-1 of host
origin, mediates the early leukocyte infiltrate (Fig. 2A). Because
the inflammatory process requires a sustained influx of leukocytes
for several days, we next assessed the number of infiltrating cells
5 d after injection. Indeed, in comparison with day 1, the number
of infiltrating cells increased ∼2-fold in Matrigel plugs (BD
Biosciences) containing hypoxic supernatants (Fig. 2B). Surprisingly, neutralization of IL-1a or injection of supernatants into IL1a KO host mice resulted in a noticeable reduction in the number
of infiltrating leukocytes. However, neutralizing IL-1b or injection
of Matrigel plugs (BD Biosciences) into IL-1b KO mice resulted
in a more significant reduction in the number of infiltrating cells
(Fig. 2B). These results indicate that for initiation of inflammation,
IL-1a is essential, whereas for propagation of the inflammatory
response, IL-1b plays a dominant role.
Next, we characterized the cells that infiltrate into Matrigel plugs
(BD Biosciences) containing hypoxic supernatants 24 h and 5 d
following injection. The vast majority of infiltrating cells was of
myeloid lineage with a phenotype of SSChigh/CD11b+/GR1high
neutrophils and or CD115+/CD11b+/Ly6Chigh monocytes. However, on day 5, the infiltrating myeloid cell populations were dramatically altered, and mainly CD11b+/Ly6Cdull/F4-80high mature
macrophages, rather than monocytes, were observed, concomitant
with neutrophil disappearance (Supplemental Fig. 2). Addition of
anti–IL-1a Abs to Matrigel (BD Biosciences) containing hypoxic
supernatants or injection into IL-1a KO mice largely reduced
neutrophil recruitment on day 1 (Fig. 2C), yet only marginally
affected the accumulation of monocytes and macrophages. For
example, neutrophils consisted 46% of the total infiltrate in WT
mice, 6.8% in IL-1a KO, and IL-1b KO mice 38.7%. Upon
blocking IL-1b, either by addition of anti–IL-1b Abs to Matrigel
(BD Biosciences) or by injection of supernatants to IL-1b KO
mice, neutrophil recruitment did not change on day 1 (Fig. 2C), yet
we observed significantly reduced macrophage recruitment into
Matrigel plugs (BD Biosciences) on day 5 compared with WT
mice (Fig. 2D). Taken together, these results suggest that IL-1a
released from hypoxic cells initiates inflammation, whereas hostderived IL-1a and, more pronouncedly, IL-1b are needed to potentiate and maintain the inflammatory process.
FIGURE 2. IL-1a initiates inflammation by promoting neutrophil recruitment, whereas IL-1b recruits macrophages. Supernatants of normoxic
or hypoxic BD7 keratinocytes injected as such, with neutralizing Abs
against IL-1a or IL-1b into WT, or as such into IL-1a or IL-1b KO mice.
A, Total number of infiltrating cells in Matrigel plugs (BD Biosciences)
after 24 h. B, Total number of infiltrating cells in Matrigel plugs 5 d following injection. C, Flow cytometry analysis of infiltrating cells in
Matrigel plugs on day 1. Numbers indicate the percentage of GR1high
(neutrophils) or Ly6Chigh (monocytes) from CD11b+ cells. Mean 6 SEM
of neutrophil recruitment from four experiments is presented in lower right
panel. D, Flow cytometry analysis of infiltrating cells in Matrigel plugs on
day 5. Numbers indicate the percentage of F4/80high cells from CD11b+
cells. Mean 6 SEM of macrophage recruitment from four experiments is
presented in lower right panel. Data in A and B are from three experiments
(n = 9). Results represent the mean 6 SEM. *p , 0.05, ***p , 0.001,
compared with the WT group.
The Journal of Immunology
4839
tory process. For this purpose, infiltrating cells were recovered
from Matrigel plugs (BD Biosciences) containing hypoxic supernatants 24 h and 5 d following injection into WT mice and stained
for intracellular IL-1 molecules and lineage markers. On day 1, IL1a was expressed in 15% of the cells that were Gr1hi/CD11bhi
neutrophils. Expression of IL-1b was only marginal in infiltrating
cells into Matrigel plugs (BD Biosciences) on day 1; it was found
only in ∼0.8% of infiltrating cells, all Gr1hi/CD11bhi neutrophils
(Fig. 3B). On day 5, IL-1a in CD11b-positive myeloid cells was
almost nonexistent. In contrast, the expression of IL-1b was
markedly elevated in day 5 Matrigel plug (BD Biosciences)infiltrating cells, reaching up to 3% of the cells, all macrophages
(Fig. 3A). To corroborate these results, frozen sections of Matrigel
plugs (BD Biosciences) containing hypoxic supernatants were
costained for the IL-1 and CD11b molecules. On day 1, high levels
of IL-1a, and also IL-1b, were found in CD11b-positive cells (Fig.
3B). Consistent with the flow cytometry results (Fig. 3A), low
levels, if any, of IL-1a were found on day 5, whereas IL-1b was
abundantly expressed in CD11b-positive myeloid cells at this time.
agation of IL-1a–induced inflammation. Hence, Matrigel (BD
Biosciences) containing supernatants of hypoxic BD7 cells were
injected into WT and caspase-1 KO mice. Matrigel (BD Biosciences) was also injected into IL-18BP transgenic mice because
caspase-1 is required for the processing of IL-18, which is known
Recruitment of macrophages during sterile inflammation is
caspase-1 dependent
In contrast to IL-1a, which is active as a precursor, IL-1b must be
cleaved by caspase-1 (also known as IL-1–converting enzyme) on
the inflammasome prior to its secretion (32). However, several
studies suggested that IL-1b might also be activated and processed
in a caspase-1–independent manner by extracellular proteases,
particularly in cases in which neutrophils are abundant (33). We
thus assessed the role of caspase-1 in sterile inflammation, relevant to the later phase of macrophage recruitment and the prop-
FIGURE 4. Recruitment of macrophages is caspase-1 dependent. A,
Total infiltrating cells on day 1 in Matrigel plugs (BD Biosciences) containing supernatants of hypoxic BD7 cells. B, Total infiltrate cell number of
Matrigel plugs, containing supernatants of hypoxic BD7 cells, on day 5. C,
Flow cytometry analysis of infiltrating cells in Matrigel plugs on day 5.
Numbers indicate the percentage of F4-80+/CD11b+ cells. Results shown
are from a three experiment (n = 9). *p , 0.05, ***p , 0.001 versus WT
group.
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FIGURE 3. IL-1a and IL-1b are differentially
expressed by myeloid cells. A, Intracellular staining
of IL-1a and IL-1b in total cell infiltrate in Matrigel
plugs (BD Biosciences) containing supernatants of
hypoxic BD7. B, Frozen sections of Matrigel plugs
containing supernatants of hypoxic BD7 costained
for CD11b (green) and IL-1a (yellow) or for CD11b
(green) and IL-1b (yellow). Photomicrographs and
FACS analysis, shown in this figure, are from a representative experiment out of three performed (original magnification 3400).
4840
for its ability to mediate inflammation in ischemic disorders (34).
As shown in Fig. 4A, in 24 h Matrigel plugs (BD Biosciences), the
number of infiltrating cells was comparable in all experimental
groups. However, on day 5, a marked reduction in inflammatory
cell number was observed in Matrigel plugs (BD Biosciences) in
caspase-1 KO mice. A less significant reduction was observed in
plugs in IL-18BP transgenic mice, in which IL-18 activity is
neutralized by the binding protein (Fig. 4A). Similar to the results
obtained in IL-1b KO mice, reduced levels of macrophages were
found in plugs from caspase-1 KO mice (Fig. 4C). Therefore,
caspase-1–processed IL-1b plays a major role in the propagation
of sterile inflammation, possibly also assisted by additional factors, such as IL-18 cleavage.
IL-1a and IL-1b mediate different myeloid cell infiltrations:
precursor IL-1a induces neutrophil infiltration, whereas
mature IL-1b recruits macrophages
Our data (Fig. 1B, middle panel, Supplemental Fig. 3B) as well as
other reports strongly indicate that IL-1a is mainly present as
STERILE INFLAMMATION IS IL-1 DEPENDENT
a precursor (22) rather than in its processed form. In addition, even
after processing to obtain mature IL-1a, most of the protein still
exists in its precursor form (35). It has been suggested that mature
IL-1a and IL-1b in their native or recombinant forms exert the
same biological activities. However, it is not known whether the
precursor of IL-1a, which is the abundant form of this cytokine in
many cell types, has the same biological activities as the mature
forms of IL-1a or IL-1b. Furthermore, as shown above, IL-1a and
IL-1b are expressed at different phases of the inflammatory response and are derived from different infiltrating cells, suggesting
that they may have distinct biological roles. To approach the latter,
we decided to examine the activity of the IL-1 molecules in their
native forms as produced by cells. Thus, HEK-293T cells were
transfected with constructs of either the precursor form of IL-1a
or the mature form of IL-1b fused to GFP. Confocal microscopic
analysis indicated typical cellular compartmental localization of
precursor IL-1a in the nucleus, whereas IL-1b was found in the
cytosol (Fig. 5A). To verify that each clone expressed only one
Downloaded from http://www.jimmunol.org/ by guest on June 15, 2017
FIGURE 5. IL-1a and IL-1b recruit different myeloid infiltrating cells into Matrigel plugs (BD Biosciences). A, Confocal microscopy of HEK-T293 cells
transfected with either GFP alone, GFP fused to the precursor of IL-1a (precIL-1a GFP), or to mature IL-1b (matIL-1b GFP) (original magnification
3400). B, Western blot analysis of lysates from HEK-T293–transfected cells stained with anti–IL-1a, anti–IL-1b, or anti-GFP Abs. C, Flow cytometry
analysis of cell infiltrate in Matrigel plugs containing lysates of transfected cells 24 h after injection. Numbers indicate the percentage of CD11b-gated cells.
Mean 6 SEM of neutrophil and macrophage recruitment from three different experiments is presented in right panels. *p , 0.05 versus GFP group.
The Journal of Immunology
Discussion
IL-1a has recently been described as a major alarmin molecule
involved in the initiation of sterile inflammation, as evidenced by
early neutrophil infiltration into affected sites. This has been
shown in experimental models of peritonitis (9, 10, 36) and in
a model of inflammation induced in Matrigel plugs (BD Biosciences) supplemented with products of damaged tissue, which
we have established in our laboratory (26). IL-1a, IL-33, and
HMGB1 belong to a family of dual-function cytokines, which can
be located in the nucleus of cells where they perform regulatory
functions, or they can be released into the microenvironment and
act as alarmins, inducing inflammation (11–13). We have shown
that intracellular IL-1a, which is constitutively expressed at low
levels in epithelial or mesenchymal cells, is released upon cell
necrosis, whereas in apoptotic cells, it avidly binds to the chromatin and is not released into the microenvironment and thus
possibly acts to restrain inflammation. To induce necrotic cell
death, hypoxia was chosen, as it represents one of the major forms
of stress and is involved in ischemia-induced tissue damage,
Downloaded from http://www.jimmunol.org/ by guest on June 15, 2017
type of the IL-1 molecules, lysates from the transfected cells were
subjected to Western blot analysis using specific Abs. Indeed,
immunoblotting of cell lysates using anti–IL-1a Abs resulted in
two bands only in cells transfected with the IL-1a construct: one
major band of 59 kDa, which represents the precursor form of IL1a fused to GFP, and a weaker band at 45 kDa, representing the
processed form of IL-1a fused to GFP (Fig. 5B, top panel).
Blotting with anti–IL-1b Abs resulted in a single 45-kDa band
only in cells transfected with the mature IL-1b construct, representing mature IL-1b fused to GFP (Fig. 5B, middle panel). The
Western blot was also performed with anti-GFP Abs that verified
the expression patterns of the vectors in control and IL-1–transfected cells (Fig. 5B, bottom panel).
Next, we assessed whether cell-derived IL-1a and IL-1b in the
above-described experimental system recruit the same or different
inflammatory cells. Thus, Matrigel (BD Biosciences) was mixed
with lysates from either GFP-transfected cells, precursor IL-1a–
transfected cells, or mature IL-1b–transfected cells and injected
into WT mice. After 24 h, plugs were removed, and the infiltrating
cell populations were analyzed by flow cytometry. Although GFPcontaining lysates had a minute effect on cell recruitment, the
lysates of both IL-1–GFP-containing Matrigels (BD Biosciences)
displayed massive cell recruitment (Supplemental Fig. 4). As
shown in Fig. 5C, Matrigel (BD Biosciences) mixed with lysates
from IL-1a–transfected cells recruited significantly higher levels
of SSChigh/CD11b+/GR1high/Ly6Ghigh neutrophils compared with
Matrigel (BD Biosciences) containing GFP- or IL-1b–transfected
cells. In contrast, Matrigel (BD Biosciences) mixed with lysates
from IL-1b–transfected cells induced massive recruitment of
SSClow/CD11b+/CXCR4+/F4-80+ inflammatory monocytes, whereas
Matrigel (BD Biosciences) mixed with lysates from GFP- or IL1a–transfected cells recruited only basal levels of these cells (Fig.
5C). We verified that the recruitment of these populations was
dependent on IL-1 signaling by injecting into IL-1R KO mice
(Supplemental Fig. 4). These results indicate that the different
molecules of IL-1 recruit distinct populations of myeloid cells.
4841
Cell lysates of lung, liver, heart, and kidney trigger neutrophil
recruitment in an IL-1a–dependent manner
Finally, to confirm that IL-1a–mediated sterile inflammation, as
induced by necrotic keratinocytes, is of physiologically relevance
to tissue damage in vivo, cell lysates from different representative
organs, including heart, kidney, liver, and lungs, were assessed
for their inflammation-inducing potential. Thus, tissue cells were
separated, isolated, and lysates were prepared from them. We
chose this approach because IL-1a is mainly intracellularly located, and we aimed to assess the role of tissue cell-derived IL-1a
in the induction of inflammation, trying to minimize the effects of
extracellular proteins. Subsequently, equal amounts of lysates
from single-tissue cell suspensions, as calibrated by protein levels,
were mixed with Matrigel (BD Biosciences) and injected into WT
mice, with anti–IL-1a–neutralizing Abs or with control IgG. After
24 h, Matrigel plugs (BD Biosciences) were removed, and infiltrating cells were counted and analyzed by flow cytometry. The
results show that cell products originating from all tissues examined were able to recruit infiltrating cells into Matrigel plugs (BD
Biosciences), and the infiltration was significantly reduced upon
IL-1a neutralization (Fig. 6A). Furthermore, flow cytometry
analysis showed a marked reduction in CD11b+/GR1high/Ly6Ghigh
neutrophils following IL-1a neutralization compared with the
isotype IgG control (Fig. 6B). Thus, these results show that IL-1a
serves as an alarmin, initiating sterile inflammation in different
tissues, such as skin, heart, kidney, liver, or lungs following necrosis.
FIGURE 6. The recruitment of neutrophils depends on IL-1a released
from necrotic tissues. A, Total cell number infiltrating on day 1 into
Matrigel plugs (BD Biosciences) containing 20 mg of tissue extracts, with
anti–IL-1a neutralizing Abs or with control Abs. Shown are data from
three experiments (n = 9). Results represent the mean value 6 SEM. B,
Flow cytometry analysis of infiltrating cells after 24 h. Numbers represent
GR1high/Ly6Ghigh (neutrophils) from CD11b-gated cells. ***p , 0.001
versus IgG CT groups.
4842
also be possible that other products of damaged tissue contribute
to the inflammatory response of cell-derived IL-1. However, the
dominant role of the IL-1 molecules in induction and maintenance
of sterile inflammation is demonstrated by the reduction of cell
recruitment to background levels upon IL-1 neutralization or using
IL-1 KO mice. We assume that the unique effects of the IL-1
molecules on the inflammatory response are through a cascade
of cytokines/chemokines that they induce. As such, it may be that
different cell-associated IL-1 molecules may induce a different
array of molecules, which may fine-tune the inflammatory
responses. Preliminary results in this direction demonstrate that
cytokines are induced in a stronger manner by the precursor of IL1a than by mature IL-1b and that G-CSF, which has been involved in neutrophil infiltration, is induced only by the precursor
of IL-1a (P. Rider, unpublished observations). The role of cellassociated IL-1a in inducing sterile inflammation is a general
phenomenon, because lysates from heart, kidney, liver, and lungs
induced an inflammatory response in an IL-1a–dependent manner.
These results are in accordance with our previous results in which
necrotic cells lacking IL-1a (lysates of primary fibroblasts from
IL-1a KO mice or from WT mice treated with anti–IL-1a Abs)
failed to induce this early inflammatory response. It is notable that
relatively small quantities of cell lysates of the different organs
were used to induce inflammation, and we also assured that the
cell content, rather than extracellular proteins, was used to induce
inflammation. Our preliminary results also indicated the role of
IL-1a in skin wound healing, which is retarded in IL-1a KO mice
(O. Guttman, unpublished observations). This further substantiates
the results of other studies showing that cell-associated IL-1a is an
alarmin of sterile inflammation (9, 10, 36) and supports models of
tissue damage to the liver (45, 46), heart (47), kidney (48), and
lungs (49), showing dependence on IL-1 signaling. It has to be
emphasized that the differential role of IL-1a and IL-1b described
in this study is relevant to sterile inflammation, and, in other
models, the specific action of the IL-1 molecules may differ.
Overall, our results show a sequential action for IL-1a and IL-1b
in sterile inflammation. Initially, the inflammatory response is
induced by IL-1a derived from damaged cells that possibly
induces host-derived IL-1a that helps to maintain the response.
IL-1b is important for the propagation of the inflammatory response. In addition, we have delineated specific functions of cellassociated precursor IL-1a that are distinct from mature IL-1b;
thus, precursor IL-1a recruits neutrophils, whereas mature IL-1b
recruits macrophages. These results may lead to novel approaches
to intervene in processes in which there is infiltration of specific
myeloid cells in tissue damage and inflammation.
Disclosures
The authors have no financial conflicts of interest.
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