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Prepublished online as a Blood First Edition Paper on August 15, 2002; DOI 10.1182/blood-2002-03-0921.
 Previous Article | Table of Contents | Next Article 
Blood, 1 January 2003, Vol. 101, No. 1, pp. 210-215
IMMUNOBIOLOGY
Early macrophage influx to sites of cutaneous granuloma formation
is dependent on MIP-1 / released from neutrophils recruited by
mast cell-derived TNF
Esther von Stebut,
Martin Metz,
Genevieve Milon,
Jürgen Knop, and
Marcus Maurer
From the Department of Dermatology, University-Hospital
Mainz, Germany; and Unite d'Immunophysiologie et
Parasitisme Intracellulaire, Institut Pasteur, Paris,
France.
 |
Abstract |
Macrophages (M ) play a crucial role in the development of
cutaneous granulomas (CGs) initiated by foreign bodies or invasive microorganisms. However, little is known about how M are recruited to sites of CG formation. To test whether mast cells (MCs) contribute to early M recruitment to developing granulomas, CGs were induced in MC-deficient KitW/KitW-v mice
by injection of polyacrylamide gel (PAG).
KitW/KitW-v mice as well as mice
deficient in the MC product TNF exhibited markedly reduced M
numbers in CGs. M recruitment was restored in
KitW/KitW-v mice reconstituted with
MCs from Kit+/+ or
TNF+/+, but not from TNF / mice.
MC-TNF-dependent M influx required prior recruitment of
MIP-1/ -producing neutrophils (PMNs), as PMN depletion before induction of CGs completely inhibited M influx, which was restored after reconstitution with PMN supernatants. These findings indicate that M recruitment to cutaneous PAG- induced granulomas is the result of a sequence of inflammatory processes initiated by MC-derived TNF followed by PMN influx and MIP-1a/ release.
(Blood. 2003;101:210-215)
© 2003 by The American Society of Hematology.
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Introduction |
Mononuclear phagocytes (M) are crucial effector
cells in the induction of protective cutaneous immune responses to
infections by (1) microbes such as Leishmania
major and Mycobacterium leprae1 or
(2) foreign bodies such as polyacrylamide gel (PAG). M-mediated protection from such intracellular microorganisms includes
phagocytosis, recruitment of other proinflammatory leukocytes (ie,
neutrophils [PMNs], eosinophils, and lymphocytes),1,2
and, most importantly, development of cutaneous granulomas (CGs) aimed
at clearing or restricting the growth of microorganisms at sites of
infection.1,3 Failure to recruit M and PMNs in
microbial infection results in impaired granuloma formation associated
with greatly impaired host defense and systemic disease.4
Despite the great importance of M in granulomatous
inflammation,2 little is known about the mechanisms
regulating their recruitment to developing cutaneous granulomas.
To better characterize the chain of inflammatory processes preceding
M-dependent CG formation and to elucidate potential mechanisms
involved in M recruitment, we have investigated PAG-induced granulomatous inflammation in the absence of mast cells (MCs). MCs are
well known to initiate and orchestrate inflammatory skin responses to
allergens and pathogens. We and others have shown that early activation
of MCs is necessary for protective immune responses to bacterial
infection in the context of acute septic peritonitis.5-8
In this setting, impaired MC activation, such as in complement
(C3)-deficient mice, correlates with markedly reduced (1) release of
TNF, (2) PMN recruitment, (3) clearance of bacteria, and,
consequently, (4) survival.7 Furthermore, MCs have been
shown to contribute to acute inflammatory monocyte recruitment by
releasing MCP-1 or histamine in allergic asthma as well as after the
implantation of biomaterial.9,10 However, the role of MCs
in more slowly developing immune responses such as cutaneous
granuloma formation has not yet been investigated.
Here, we assessed early CG formation in MC-deficient mice, using the
model of PAG-induced granulomatous inflammation. We demonstrate that
MCs are required to initiate a sequence of inflammatory processes resulting in M recruitment to developing CGs: MC-derived TNF is
necessary to recruit PMNs, which control M influx by releasing M-attracting chemokines, including macrophage inflammatory protein-1 (MIP-1) / and MIP-2.
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Materials and methods |
Animals
C57BL/6 mice, genetically MC-deficient
WBB6F1-KitW/KitW-v
(KitW/KitW-v) mice, and congenic wild-type
WBB6F1-Kit+/+
(Kit+/+) mice were purchased from The Jackson
Laboratory (Bar Harbor, ME). TNF/ mice of a mixed
129/Sv × C57BL/6 genetic background were bred at the
Department of Dermatology, Mainz.11 Mice were kept in community cages at the Animal Care Facilities at the University of
Mainz. All animal care and experimentation were conducted in accordance
with current federal, state, and institutional guidelines.
CG model
CGs were elicited by subcutaneous or intradermal injection of
polyacrylamide gel (PAG) as previously described.12-14
Briefly, PAG was prepared by addition of Biogel P-100 (BioRad,
München, Germany) to phosphate buffered saline (PBS; 35 mg/mL). PAG was injected subcutaneously into the neck area in
volumes of 1 mL. Initial experiments with C57BL/6 mice confirmed that
the cellular infiltrate in  5-day-old granulomas consisted
exclusively of PMNs and M as assessed by fluorescence-activated
cell-sorter (FACS) analysis.15 In some
experiments, depleting anti-mouse neutrophil rat mAb clone NIMP-R14
(100 µg/mouse)16 or clone 7/4 (200 µg/mouse) (Serotec,
Hamburg, Germany) or isotype control rat mAb were injected intravenously.
Phenotyping of leukocytes recovered from PAG-induced granulomas
Leukocytes were harvested from early PAG-induced granulomas and
separated from PAG by filtering through a 70-µm filter in cold PBS.
Cells were counted, stained for surface Ag expression,14 and analyzed using a FACScan flow cytometer equipped with CellQuest software (Becton Dickinson, Heidelberg, Germany). The antibodies used
were as follows: biotin-conjugated F4/80 and fluorescein isothiocyanate
(FITC)-conjugated anti-neutrophil mAb (7/4) were obtained
from Caltag (Hamburg, Germany); anti-CD16/CD32 (2.4G2), anti-I-Ab (2G9), anti-CD3 (145-2C4), anti-CD4 (L3T4),
anti-CD8 (53-6.7), CD11b (M1/70), Gr-1 (RB6-8C5), and anti-NK1.1
(PK136) were purchased from PharMingen (Hamburg, Germany) as biotin- or
phycoerythrin (PE)-modified mAb; PE-streptavidin was from Tago
(Burlingame, CA).
Preparation of MCs
MC-deficient skin was reconstituted with peritoneal MCs as both
peritoneal and skin MCs are connective tissue type MCs (CTMCs) and
share virtually identical phenotypes.8 In brief, the
peritoneal cavity was lavaged with 0.9% NaCl, and cell suspensions
were stained for MCs by Kimura stain and enumerated. CTMCs were
enriched to  95% purity from peritoneal lavage suspension by
several gradient centrifugation steps using 23%
metrizamide.17 Viability of CTMCs was 90%
as assessed by trypan blue exclusion.
Cutaneous MC reconstitution of
KitW/KitW-v mice
The MC deficiency of
KitW/KitW-v mice (6-8 weeks old) was
corrected selectively and locally by injection of CTMCs.8
CTMCs (1 × 106 in 100 µL 0.9% NaCl) were injected
intradermally, and mice were used for experiments, together with sex-
and age-matched MC-deficient KitW/KitW-v and
Kit+/+ mice, 48 hours after adoptive transfer.
MCs were injected into shaved neck skin covering an area of
about 1 cm2. Reconstitution of cutaneous MC
populations was confirmed by histomorphometric analysis of
paraffin-embedded, Giemsa-stained sections of injected skin 48 hours
after the injection.8
MC activation assays
The extent of MC degranulation in PAG-injected skin was assessed
as described previously.18 Biopsies were taken 60 minutes after intradermal injection of 0.1 mL PAG, and sections were examined by an investigator blinded to the experimental design. MC degranulation was assessed by quantitative histomorphometry (at
1000×).18 A minimum of 100 MCs in 5 sections/mouse per treatment group were examined. Measurements of
serotonin (5-hydroxytryptamine [5-HT]) release in vitro were
performed as previously described.17 Briefly, peritoneal
MC suspensions were incubated with 2 µCi (0.074 MBq)[3H]5-HT (Perkin Elmer, Freiburg, Germany)
for 2 hours at 37°C, washed, stimulated with PAG or PBS for 15 minutes, and the percentage of total 5-HT release was calculated.
Reconstitution of CGs with PMN supernatants and RNAse protection
assay
PMNs were isolated from 6- and 12-hour-old granulomas by filter
separation. PMNs were further enriched by negative depletion of M
using a 2-hour plastic adherence step; > 99% of cells in PMN
preparations routinely showed positive antineutrophil (clone 7/4)
staining as demonstrated by FACS analysis. Chemokine production was
determined by enzyme-linked immunosorbent assay (ELISA) (R&D Systems, Wiesbaden, Germany) in 18-hour supernatants of
2 × 106 PMN/mL RPMI/5% fetal calf serum (FCS)
supplemented with glutamine, penicillin/streptomycine, and nonessential
amino acids.14 PMN supernatants for reconstitution
experiments were generated by plating 5 × 106 PMN/mL in
RPMI/5% FCS in 6-well plates, and cell-free supernatants were
harvested after 48 hours. As a control, RPMI/5% FCS was treated similarly. Supernatants were assayed by ELISA for their chemokine content and were stored at  20°C until used for reconstitution experiments. Biogel granulomas were reconstituted locally twice daily
with either media alone or PMN supernatants (0.5 mL/granuloma) or PMN
supernatants that had been preincubated with 10 µg/mL anti-MIP-1 and 0.1 µg/mL anti-MIP-1 (R&D Systems) for 30 minutes at 20°C (Ab amounts were about 4-fold of a concentration calculated to inhibit
approximately 50% of the activity found in the supernatants).
For analysis of chemokine expression, RNA from PMNs isolated from 6- and 12-hour granulomas was generated using the High Pure RNA Isolation
Kit from Roche (Mannheim, Germany). Expression of chemokines known to
be potent M attractants was assessed following the manufacturer's
protocol using the RiboQuant RNAse Protection System and template set
mCK5, both obtained from PharMingen.
Statistical analysis
All data were tested for statistical significance using the
unpaired 2-tailed Student t test or  2 test
(MC degranulation in situ) and are presented as means ± SEM.
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Results |
Recruitment of M during early granuloma formation is
MC-dependent
M recruitment to early CGs induced by subcutaneous injection of
PAG was greatly reduced in genetically MC-deficient
KitW/KitW-v mice compared with
Kit+/+ mice during the first 72 hours following
CG induction (Figure 1A). M numbers in
CGs of MC-deficient skin were reduced by 66% (0.3 ± 0.1 vs
0.9 ± 0.1 × 106, P = .001) and 46%
(3.5 ± 0.6 vs 6.6 ± 0.7 × 106,
P = .004) at 12 hours and 72 hours, respectively, compared
with skin of wild-type mice (Figure 1B). To ensure that this defect is
due to a lack of MCs in these mice, we reconstituted the skin of
KitW/KitW-v mice with
Kit+/+ CTMCs (1 × 105) prior to
the induction of granulomas, which fully restored the influx of
inflammatory M to CGs after 12 hours (Figure 1B). These data suggest
that MCs are required for normal recruitment of M to sites of CG
formation.
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| Figure 1.
Early M recruitment to PAG-induced cutaneous
granulomas is MC dependent.
The absence of MCs in KitW/KitW-v
mice results in impaired recruitment of M to early CGs
developing at sites of subcutaneous delivery of PAG. (A) Time
course of M recruitment to CGs after PAG injection in
Kit+/+ and
KitW/KitW-v mice. Data were pooled
from 8 or more mice per genotype and time point-tested in at least 4 independent experiments. (B) Migration of M to granulomas in
KitW/KitW-v mice 12 hours after
induction is repaired by reconstitution of the dermis with connective
tissue-type MCs obtained from Kit+/+ mice. Data
were pooled from 8 or more mice per genotype in 2 independent
experiments. Data in panels A and B are shown as means ± SEMs
(× 106 cells/granuloma). In 1A and 1B,
*P < .05, **P < .005,
***P < .001. (C) Characteristic surface staining with
anti-M F4/80 and anti-PMN 7/4 mAb of leukocytes recovered from 24- and 72-hour-old granulomas of normal Kit+/+ mice
revealed 2 distinct populations, namely PMNs (7/4+) and
M (F4/80+). Numbers reflect
F4/80+/7/4 cells and
7/4+/F4/80 cells in percent of total cells.
One experiment representative of at least 8 independent analyses is
shown.
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M from developing granulomas were readily identified as
F4/80+ and 7/4 cells (Figure 1C). In both
MC-deficient KitW/KitW-v mice and
normal Kit+/+ mice, M influx to CGs was
detected as early as 12 hours after PAG injection. Interestingly, the
surface phenotype of infiltrating M (eg, presence of major
histocompatibility complex class I and II, or costimulatory molecules)
was not affected by the absence of MCs (data not shown).
Recruitment of M to skin granulomas is dependent on MC-derived
TNF
To identify the mechanisms by which MCs control M recruitment
to early CGs, we first determined whether CG formation after injection
of PAG is associated with MC activation. PAG-injected skin exhibited
significantly more extensively and moderately degranulated MCs than
vehicle-treated or uninjected skin (P < .001) 1 hour after induction of granulomas in C57BL/6 mice (Figure
2A). PAG was also found to induce
substantial degranulation of isolated and highly purified CTMCs
in vitro as assessed by serotonin release assays (Figure
2B), suggesting the MCs may recruit M to developing CGs
by releasing preformed proinflammatory mediators.
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| Figure 2.
Induction of cutaneous granuloma formation by PAG
depends on release of TNF by MCs.
(A) MCs in the direct vicinity of early PAG-induced CGs
exhibit signs of profound degranulation. As assessed by quantitative
histomorphometry, the extent of MC degranulation 1 hour after PAG
injection in C57BL/6 mice was scored as "none" (< 10% of
granules exhibited staining alterations and/or exteriorization),
"moderate" (10%-50%), or "extensive" (> 50%), and
expressed as means ± SEMs (data pooled from 5 mice). (B) Unpurified (purity about 1%) and purified
(purity > 95%) peritoneal MCs of C57BL/6 mice release
serotonin after incubation with PAG. Data were pooled from 3 independent experiments and expressed as means ± SEMs. (C)
TNF-deficient mice exhibit reduced recruitment of M to sites of
CG formation after injection of PAG compared with wild-type mice. Data
were pooled from 5 or more mice per genotype and time point in 3 independent experiments. (D) Migration of M to granulomas in
KitW/KitW-v mice 12 hours after
induction is repaired only by reconstitution of the skin with
connective tissue-type MCs obtained from TNF+/+ mice, but not after
reconstitution with MCs from TNF-deficient mice. Data were pooled
from 8 or more mice per genotype in 2 independent experiments. Data in
all panels are shown as means ± SEMs. *P < .05,
**P < .005, ***P < .001.
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Since one such MC product, TNF, has been shown to regulate the
recruitment of inflammatory cells, including M,19-22 we
assessed PAG-induced CG formation in TNF/ mice and TNF+/+
mice. M recruitment to CGs was dramatically impaired in TNF/
mice as compared to TNF+/+ mice (Figure 2C). Notably, up to 7-fold
more M per granuloma were recovered in TNF+/+ mice as compared to TNF/ mice (8.9 ± 1.3 vs 1.3 ± 0.7 × 106 at
72 hours, P < .001).
Since TNF is not exclusively produced by MCs, but MCs are the only
cell type known to contain preformed TNF,19,23 we reconstituted the skin of
KitW/KitW-v mice with MCs derived
from either TNF+/+ mice or TNF/ mice, thus generating mice
that differed solely in containing MCs that could or could not release
TNF.7,24 KitW/KitW-v
mice showed normal migration of M to PAG-induced granulomas only
after reconstitution with TNF+/+ MCs (Figure 2D). Reconstitution with MCs derived from TNF/ mice did not increase M influx, indicating that release of TNF from MCs is required for normal M
recruitment to sites of CG formation.
M influx to CGs is dependent on PMNs recruited by MC-TNF
M migration to inflamed skin is preceded by the influx of large
amounts of PMNs, proinflammatory cells that have been shown to migrate
to sites of TNF release from MCs and to produce M-recruiting chemokines.25 To assess whether MC-TNF recruits M
during early CG formation directly or by inducing immigration of
M-attracting PMNs, we first assessed the kinetics of PMN recruitment
to PAG-induced granulomas in MC-, TNF-, or MC-TNF-deficient
skin. Genetically MC-deficient
KitW/KitW-v mice exhibited markedly
reduced recruitment of PMNs as compared to wild-type mice after
induction of CGs by PAG injection (Figure 3A). PMN numbers in 6- to 72-hour-old CGs
were significantly and up to 70% lower as compared to wild-type mice
at all time points studied. The absence of MCs did not affect the time
course of PMN influx. Adoptive transfer of CTMCs to MC-deficient skin
prior to injection of PAG restored normal recruitment of PMNs to CG (Figure 3B). PMN numbers also were greatly reduced in TNF-deficient mice as compared to TNF+/+ mice in up to 5-day-old granulomas (Figure 3C). PMN recruitment was completely restored in
KitW/KitW-v mice reconstituted with
TNF+/+ MCs, while reconstitution with TNF-deficient MCs did
not correct impaired PMN influx in these mice. These observations
indicate that MC-derived TNF contributes not only to M
recruitment, but also to PMN recruitment to sites of CG formation.
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| Figure 3.
Neutrophil recruitment to PAG-induced granulomas is
dependent on MC-derived TNF.
(A) Time course of PMN influx after injection of PAG in
Kit+/+ and KitW/KitW-v
mice. Data were pooled from 10 or more mice per genotype and time
point-tested in at least 7 independent experiments. (B) Impaired
recruitment of PMNs to CGs in
KitW/KitW-v mice is MC dependent.
The dermis of KitW/KitW-v mice was
reconstituted with connective tissue-type MCs from normal
Kit+/+ mice before PAG injection, and PMN numbers were
determined 12 hours after injection of PAG. Data were pooled from 8 or
more mice per genotype in at least 4 independent experiments. (C) PMN
recruitment in cutaneous granuloma formation is strongly dependent on
the presence of TNF. Numbers of infiltrating PMNs were determined in
PAG-injected skin of mice deficient in TNF and in wild-type mice.
Data were pooled from 5 or more mice per genotype and time point in 3 independent experiments. (D) PMN recruitment was fully restored in skin
of TNF+/+ MC-reconstituted
KitW/KitW-v mice. Before PAG
injection, the dermis of KitW/KitW-v
mice was reconstituted with MCs from TNF+/+ mice or TNF-deficient
mice, and numbers of PMNs were assessed 12 hours thereafter. Data were
pooled from 8 or more mice per genotype in 2 independent experiments.
All data in Figure 3 are shown as means ± SEMs
(× 106 cells/granuloma). For all panels in Figure
3, *P < .05, ***P < .005,
***P < .001.
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To determine if MC- and TNF-dependent recruitment of PMNs is
involved in the regulation of M recruitment to CGs, C57BL/6 mice were depleted of PMNs by a single intravenous injection of the mAb
NIMP-R14 or mAb 7/4 1-3 hours before induction of CG. NIMP-R14 has been
shown to eliminate PMNs from the peripheral blood and bone marrow of
mice for 2-3 days without affecting other leukocyte populations of the
myeloid lineages.16 PMNs were found to be virtually absent
in PAG-induced CGs after injection of NIMP-R14, whereas treatment with
the isotype control mAb had no effect compared with untreated animals
(Figure 4A). Notably, the numbers of
F4/80+ M were reduced by 88% and 93% at 24 hours and
72 hours, respectively, after depletion of PMN (Figure 4B), indicating
that PMN recruitment is essential for normal M migration to sites of
CG formation.
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| Figure 4.
PMN influx is required for M recruitment during early
cutaneous granuloma formation.
C57BL/6 mice pretreated with PMN-depleting mAb NIMP-R14 (intravenously,
100 µg, 1 hour) exhibit virtually no recruitment of M
to sites of PAG-induced CG formation compared with control Ab-treated
mice. Numbers of PMNs (A) and M (B) per CG were assessed 24 hours
and 72 hours after injection of PAG. Data were pooled from 5 or more
mice per treatment group and time point in 2 independent experiments.
Data are shown as means ± SEMs (106 cells/granuloma).
***P < .001. (C) PMNs were isolated from CGs 12 hours
after injection of biogel and were stained for anti-neutrophil Ab
clone 7/4 (black area, anti-neutrophil Ab; white area, isotype
control). Purity of isolated PMNs was determined to be > 99%; one
experiment of at least 5 is shown. In panel D, groups of 3 C57BL/6 mice
were depleted from PMNs using mAb 7/4 (100 µg intravenously, at 6
hours and +18 hours), and CGs were reconstituted locally twice daily
with 0.5 mL/granuloma of either vehicle alone or PMN supernatant
(PMN-SPNT). M numbers were determined after 48 hours and are
expressed as means ± SEMs (106 cells/granuloma).
**P < .005. Data are representative of 3 independent
experiments.
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Soluble mediators released by PMNs rather than structural/molecular
changes induced by PMN migration may regulate M recruitment to CGs.
To test this hypothesis, we reconstituted CGs in PMN-depleted mice with
supernatant derived from PMNs or media alone (Figure 4D). Supernatants
from PMNs were generated in RPMI/5% FCS for 48 hours, and purity of
isolated PMNs was determined to be > 99% using FACS analysis and
staining with anti-neutrophil Ab 7/4 (Figure 4C). Local injection of
PMN supernatants twice daily restored > 60% of M influx to
sites of CG formation, whereas vehicle alone (media + FCS) did not
repair M influx at all.
MIP-1/ released by PMNs are responsible for M
recruitment to CGs
To identify the soluble mediators released by PMNs, we analyzed
cytokine release and chemokine expression of PMNs isolated from either
6- or 12-hour-old granulomas (Figure 5A).
While we found no significant production of TNF or other cytokines
(interleukin-4 [IL-4], IL-12p40, interferon- [IFN])
by PMNs when assessing the supernatants by ELISA (data not shown),
RNAse protection assays revealed strong expression of MIP-1,
MIP-1, and MIP-2 by PMNs. The C-C chemokines MIP-1 and MIP-1
are strong chemotactic signals for M (compare
Luster26), whereas MIP-2, a C-x-C chemokine and the murine
homolog for IL-8, is known to recruit PMNs themselves as well as T
lymphocytes.26 Therefore, our data suggest that PMN-derived MIP-1 and MIP-1 are responsible for M recruitment to CGs. We next assayed 18-hour-old supernatants from PMNs for protein
content by ELISA (Figure 5B). PMNs isolated from CGs
predominantly released MIP-1, although we also detected some
MIP-1 activity in the supernatants. Both chemokines were
up-regulated in TNF-treated PMNs within 18 hours (Figure 5B).
Finally, we attempted to inhibit the activity of MIP-1/ in
PMN-derived supernatants used for reconstitution experiments by
preabsorption with neutralizing antibodies. The 48-hour PMN
supernatants used for reconstitution experiments contained 3.7 ± 1.9 pg/mL MIP-1 and 197.4 ± 47 pg/mL MIP-1, whereas
RPMI/5% FCS alone was negative for the chemokines by ELISA. As
demonstrated in Figure 5C, reconstitution of CGs with PMN supernatants
treated with anti-MIP-1 and anti-MIP-1 inhibited about 60% of
supernatant-induced recruitment of M to CGs.
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| Figure 5.
MIP-1/ released by PMNs is responsible for M
influx into cutaneous granulomas.
(A) Expression of the chemokines MIP-1, MIP-1, and MIP-2 by PMNs
from early CGs (6 hours and 12 hours old) was found using the RNAse
protection assay (PharMingen). One of 2 experiments with similar
results is shown. Lane 1 shows the template set; lanes 2 and 3, PMN
RNA; lane 4, control RNA provided by the manufacturer. (B)
Production of MIP-1 and MIP-1 was determined in supernatants of
2 × 106 PMNs in fully supplemented media after 18 hours.
Chemokine production was enhanced in TNF-treated PMNs (n = 5). In
panel C, groups of 3 C57BL/6 mice were depleted of PMNs using mAb 7/4
as shown in Figure 4D, and CGs were reconstituted locally twice daily
with either vehicle alone or PMN supernatant (PMN-SPNT) or PMN-SPNT
that was pretreated with anti-MIP-1 and anti-MIP-1 for 30 minutes. M numbers were determined after 48 hours and expressed as
means ± SEMs (106 cells/granuloma).
*P < .05. Data are pooled from 6 mice per group from 2 independent experiments.
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Discussion |
MCs are ideally suited to initiate protective immune responses
against invading pathogens because (1) MCs are preferentially located
at host/environment interfaces (upper dermis in the skin, gut lamina
propria, airways' subepithelium), (2) they produce a large array of
proinflammatory mediators, many of which are stored within cytoplasmic
granules and are released within minutes after activation, and (3) MCs
have receptors that can recognize microbial molecules (including
complement receptors toll-like receptors, and CD48) and can,
therefore, be activated by pathogens via multiple mechanisms,
including fimbrial proteins, toxins, and the proteolytic fragments of
complement components.19 Indeed, MCs have been reported to
be required for the induction of immediate host defense reactions in
various models of acute bacterial infections.5-7 Here, we
show for the first time that MCs also are required to elicit normal
chronic and more slowly developing cellular responses. Indeed, the
normal succession of key events in slowly developing immune responses
such as CG formation is induced by and dependent on initial MC
activation. Specifically, early MC degranulation is needed to elicit
recruitment of M, a hallmark feature of granulomatous inflammation
(compare Murray3), which is required for the development of CGs. Our findings suggest that MCs facilitate normal chronic granulomatous inflammatory processes by inducing the following chain of
events: release of TNF from MCs promotes influx of PMN, which
release M-recruiting chemokines (such as MIP-1/, MIP-2), which
in turn result in the recruitment of M.
The early steps of granuloma development are tightly modeled in
leukocyte recruitment to lesions in cutaneous leishmaniasis, where the
initial inflammatory phase of granuloma development is characterized by
the sequential influx of PMNs, eosinophils, and M to parasite-loaded
skin.2 In leishmaniasis, extensive dermal MC degranulation
is found at sites of early infection,27 and MCs
coincubated with Leishmania major in vitro reportedly release preformed TNF within minutes,28 suggesting that
MCs and MC-derived products may modulate the host response to
Leishmania, especially during the initial phase of
infection. Our data support this hypothesis by showing that MCs
contribute significantly to the local inflammatory cutaneous reaction
that develops at sites of CG formation in response to foreign bodies
such as PAG. In future studies we will investigate in more detail if
and how MCs modulate local inflammatory responses observed after
intradermal delivery of Leishmania major.
Most of the leukocyte recruitment to CGs that we observed was TNF
dependent, stressing the essential role that TNF plays in skin
inflammation. To our surprise, the absence of TNF resulted in
reduced recruitment of M as early as 12 hours after injection of
PAG, suggesting that TNF is, at least in part, released from preformed stores. Since MCs are the only resident skin cells shown to
contain considerable amounts of preformed TNF, it is mostly likely that most of the TNF that accounts for the initial influx of
PMNs and M is released by MCs, although several other types of skin
cells are capable of producing TNF.29 At later time points after injection of PAG, the difference in inflammation between MC-deficient and normal wild-type mice was less
dramatic, perhaps reflecting TNF release or production of other
factors by cells other than MCs (including dendritic cells, resident
dermal M, keratinocytes).
Most of the MC effect on leukocyte recruitment was TNF-dependent and
did not depend on release of other MC mediators since only
reconstitution with TNF-producing MCs fully restored inflammatory cell influx to CGs in KitW/KitW-v
mice, while the adoptive transfer of TNF-deficient MCs did not improve PMNs or M recruitment in these mice. This observation is
supported by a recent report showing that inflammatory skin reactions,
including PMN influx in contact hypersensitivity, are impaired in
KitW/KitW-v mice and that these
responses were normalized only after adoptive transfer of
TNF-competent, but not TNF-deficient, MCs.24 In addition, Zhang et al provided evidence that, in vivo, MCs produce the
TNF that augments PMN emigration in the context of acute peritoneal
inflammation.30
Another remarkable finding of this study was that depletion of PMNs
resulted in a strong impairment of M recruitment to the site of CG
formation. To ensure that the mononuclear cell population was not
affected by PMN depletion, we used anti-neutrophil Ab clone NIMP-R14
for our experiments.16 In agreement with the findings of
Tacchini-Cottier et al,16 we observed a similar and
overlapping staining pattern of the monoclonal anti-neutrophil antibodies NIMP-R14 and 7/4 on PMNs derived from C57BL/6 mice. No
expression of NIMP-R14 or 7/4 by resident or recently extravasated M
was detected (Figure 1C and data not shown). The sequential accumulation of first PMNs and then M to tissues after initiation of
inflammatory processes is a phenomenon that is observed frequently in a
variety of diseases and experimental models, but the strict dependence
of M recruitment on prior PMN influx has not been previously
demonstrated. Several reports describe impaired M functions and
granuloma formation in patients with neutrophil dysfunction or
deficiency (such as agranulocytosis), for example, impaired granuloma
formation in a patient with severe granulocytopenia led to insufficient
control of fungi with severe dissemination of
Aspergillus.4 In contrast, in chronic
granulomatous disease, hyperactivation of neutrophils and increased
release of chemoattractants for M and T cells from PMNs might
contribute to poorly controlled inflammatory responses and
unstructured granuloma formation in affected
patients.31 However, further studies are warranted to
better characterize the correlation between PMNs and M recruitment in cutaneous granulomatous development and to elucidate underlying mechanisms.
In summary, we show that TNF release from activated MCs
regulates PMN influx to sites of granulomatous inflammation, which in
turn recruit M to developing cutaneous granulomas by releasing potent M-recruiting chemokines, including MIP-1 and
MIP-1. Our studies confirm and extend recently established concepts
regarding the role of MCs in host-immune responses. In addition to the
sentinel role in acute host-defense reactions against bacteria,
MCs may also be critical effector cells responsible for the
initiation and orchestration of long-lasting cell-mediated immunity.
| |
Acknowledgments |
The authors wish to thank Dr George Kollias for kindly providing
TNF-deficient mice; Drs Mark C. Udey, Yasmine Belkaid, and Helmut
Jonuleit for helpful discussions; Drs Michael Stassen and Edgar Schmitt
for help with RNAse protection assays; Drs Kerstin Steinbrink, Karsten
Mahnke, and Thomas Tüting for critically reading the manuscript;
and Elena Wiese for excellent technical assistance.
| |
Footnotes |
Submitted March 25, 2002; accepted August 6, 2002.
Prepublished online
as Blood First Edition Paper, August 15, 2002; DOI
10.1182/blood-2002-03-0921.
Parts of this work were supported by grants from the
Deutsche Forschungsgemeinschaft (DFG; Ste 833/4-1 and Ma 1909/4-1) and the Mainzer Forschungsförderungsprogramm (MAIFOR) program to E. von S. and M.M.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
Reprints: M. Maurer, Department of Dermatology,
Johannes Gutenberg-University of Mainz, Langenbeckstrasse 1, 55131 Mainz, Germany; e-mail:
maurer{at}hautklinik.klinik.uni-mainz.de.
| |
References |
1.
Schaible UE, Collins HL, Kaufmann SH.
Confrontation between intracellular bacteria and the immune system.
Adv Immunol.
1999;71:267-377[Medline]
[Order article via Infotrieve].
2.
Belkaid Y, Mendez S, Lira R, Kadambi N, Sacks DL.
A natural model of Leishmania major infection reveals a prolonged "silent" phase of parasite amplification in the skin before the onset of lesion formation and immunity.
J Immunol.
2000;165:969-977[Abstract/Free Full Text].
3.
Murray HW.
Tissue granuloma structure-function in experimental visceral leishmaniasis.
Int J Exp Pathol.
2001;82:249-267[CrossRef][Medline]
[Order article via Infotrieve].
4.
Lamendola MG.
Invasive aspergillosis: description of a case in an infant with severe granulocytopenia and endocardial fibroelastosis.
Pathologica.
1997;89:432-440[Medline]
[Order article via Infotrieve].
5.
Echtenacher B, Männel DN, Hültner L.
Critical protective role of mast cells in a model of acute septic peritonitis.
Nature.
1996;381:75-77[CrossRef][Medline]
[Order article via Infotrieve].
6.
Malaviya R, Ikeda T, Abraham SN.
Mast cell modulation of neutrophil influx and bacterial clearance at sites of infection through TNF-alpha.
Nature.
1996;381:77-80[CrossRef][Medline]
[Order article via Infotrieve].
7.
Prodeus AP, Zhou X, Maurer M, Galli SJ, Carroll MC.
Impaired mast cell-dependent natural immunity in complement C3-deficient mice.
Nature.
1997;390:172-175[CrossRef][Medline]
[Order article via Infotrieve].
8.
Maurer M, Echtenacher B, Hültner L, et al.
The c-kit ligand, stem cell factor, can enhance innate immunity through effects on mast cells.
J Exp Med.
1998;188:2343-2348[Abstract/Free Full Text].
9.
Gordon JR.
Monocyte chemoattractant peptide-1 expression during cutaneous allergic reactions in mice is mast cell dependent and largely mediates the monocyte recruitment response.
J Allergy Clin Immunol.
2000;106:110-116[CrossRef][Medline]
[Order article via Infotrieve].
10.
Tang T, Jennings TA, Eaton JW.
Mast cell mediate acute inflammatory responses to implanted biomaterials.
Proc Natl Acad Sci U S A.
1998;95:8841-8846[Abstract/Free Full Text].
11.
Pasparakis M, Alexopoulou L, Episkopou V, Kollias G.
Immune and inflammatory responses in TNF alpha-deficient mice: a critical requirement for TNF alpha in the formation of primary B cell follicles, follicular dendritic cell networks and germinal centers, and in the maturation of the humoral immune response.
J Exp Med.
1996;184:1397-1411[Abstract/Free Full Text].
12.
Fauve RM, Jusforgues H, Hevin B.
Maintenance of granuloma macrophages in serum-free medium.
J Immunol Methods.
1983;64:345-351[CrossRef][Medline]
[Order article via Infotrieve].
13.
Harris RR, Wilcox D, Bell RL, Carter GW.
The role of tissue mast cells in polyacrylamide gel-induced inflammation in mice.
Inflamm Res.
1998;47:104-108[CrossRef][Medline]
[Order article via Infotrieve].
14.
von Stebut E, Belkaid Y, Jakob T, Sacks DL, Udey MC.
Uptake of Leishmania major amastigotes results in activation and interleukin 12 release from murine skin-derived dendritic cells: implications for the initiation of anti-Leishmania immunity.
J Exp Med.
1998;188:1547-1552[Abstract/Free Full Text].
15.
Belkaid Y, Butcher B, Sacks DL.
Analysis of cytokine production by inflammatory mouse macrophages at the single-cell level: selective impairment of IL-12 induction in Leishmania-infected cells.
Eur J Immunol.
1998;28:1389-1400[CrossRef][Medline]
[Order article via Infotrieve].
16.
Tacchini-Cottier F, Zweifel C, Belkaid Y, et al.
An immunomodulatory function for neutrophils during the induction of a CD4+ Th2 response in BALB/c mice infected with Leishmania major.
J Immunol.
2000;165:2628-2636[Abstract/Free Full Text].
17.
Boesiger J, Tsai M, Maurer M, et al.
Mast cells can secrete vascular permeability factor/vascular endothelial cell growth factor and exhibit enhanced release after immunoglobulin E-dependent upregulation of Fc epsilon receptor I expression.
J Exp Med.
1998;188:1135-1145[Abstract/Free Full Text].
18.
Wershil BK, Murakami T, Galli SJ.
Mast cell-dependent amplification of an immunologically nonspecific inflammatory response: mast cells are required for the full expression of cutaneous acute inflammation induced by phorbol 12-myristate 13-acetate.
J Immunol.
1988;140:2356-2360[Abstract].
19.
Galli SJ, Maurer M, Lantz CS.
Mast cells as sentinels of innate immunity.
Curr Opin Immunol.
1999;11:53-59[CrossRef][Medline]
[Order article via Infotrieve].
20.
Chensue SW, Otterness IG, Higashi GI, Forsch CS, Kunkel SL.
Monokine production by hypersensitivity (Schistosoma mansoni egg) and foreign body (Sephadex bead)-type granuloma macrophages: evidence for sequential production of IL-1 and tumor necrosis factor.
J Immunol.
1989;142:1281-1286[Abstract].
21.
Benini J, Ehlers EM, Ehlers S.
Different types of pulmonary granuloma necrosis in immunocompetent vs. TNFRp55-gene-deficient mice aerogenically infected with highly virulent Mycobacterium avium.
J Pathol.
1999;189:127-137 |
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Abstract
Introduction