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*
Department of Clinical Immunology and
Institute of Medical Microbiology, Medical School, Hannover, Germany; and
Department of Pathology, Harvard University Medical School, Boston, MA 02115
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResults and DiscussionReferences |
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RIII. In
addition, we provided evidence for C3-independent C5aR triggering,
which may explain why the cutaneous Arthus reaction develops normally
in C3-/- mice. Furthermore, some, but not all, of
the acute changes associated with the Arthus response in the lung were
significantly more intense in normal mice than in C3-/-
or KitW/KitW-v mice,
indicating for C3- and mast cell-dependent and -independent components.
Finally, we demonstrated that C3 contributed to the elicitation of
neutrophils to alveoli, which corresponded to an increased synthesis of
TNF-
, macrophage-inflammatory protein-2, and cytokine-induced
neutrophil chemoattractant. While mast cells similarly
influenced alveolar polymorphonuclear leukocyte influx, the levels of
these cytokines remained largely unaffected in mast cell deficiency.
Together, the phenotypes of C3-/- mice and
KitW/KitW-v mice suggest
that complement and mast cells have distinct tissue site-specific
requirements acting by apparently distinct mechanisms in the initiation
of IC inflammation. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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It has long been understood that the mechanisms by which IC formation
triggers tissue injury are mainly mediated by complement activation via
the classical pathway (7). C3, the most abundant
complement protein, occupies a central position in this pathway. C3a,
which is cleaved from the -chain of the parent molecule, exerts
chemotactic activity on mast cells and activates PMN (8, 9). C3b as an essential part of the C5 convertase complex is
required for formation of C5a, the most potent chemoattractant
anaphylatoxin for PMN (10). Mice with genetic deletion or
blockade of C5aR show an impaired inflammatory response in the passive
reverse Arthus reaction of skin, peritoneum, and lung
(11, 12, 13). In addition, C3-deficient mice have a strongly
attenuated phenotype of mast cell degranulation, TNF- secretion, and
PMN infiltration in a model of cecal ligation (14). In
contrast, however, IC inflammation in the Arthus reaction develops
normally in these C3-/- mice (15, 16). It remains to be investigated whether a potential bypass of
the complement activation cascade is operative in the generation of C5a
in C3-/- mice (17, 18), thus
explaining why C3 deficiency does not disrupt the proinflammatory
properties of the complement system in general.
Recent studies using FcR-chain (lacking FcRI and FcRIII) and
FcRIII-deficient mice have revealed a critical role of activatory
FcR, especially FcRIII, in the pathogenesis of IC diseases
(19, 20, 21, 22, 23, 24). As concluded in C5aR-sufficient and
C5aR-antagonized FcRIII-/- mice, FcRIII-
and C5aR-mediated pathways are both necessary and only together
sufficient to trigger the full expression of IgG IC inflammation in
skin and lung (25). In addition, enhanced development of
both pulmonary and cutaneous Arthus reactions has been found in mice
lacking the inhibitory FcRII (16, 26). Accordingly,
FcRII-/- mice show FcRIII hyperactivation
of macrophages and mast cells (27, 28). In the lung, the
alveolar macrophage is the most prominent cell type in alveoli, and is
known to trigger the inflammatory response through the production of
various mediators (29). Mast cells have been demonstrated
as major effector cells of autoantibody- and IC-induced injury in skin
vasculitis (21, 24, 30). However, the contribution of mast
cells beside alveolar macrophages to the initiation of the pulmonary
Arthus reaction has not been analyzed.
In the present study, we examined the contribution of
FcRIII-expressing mast cells to the inflammatory response at
different tissue sites by using mast cell-deficient
WBB6F1-KitW/KitW-v
mice (31). Moreover, we analyzed the role of C3 in
relation to C5aR in the cutaneous Arthus reaction in
C3-/- mice receiving the C5aR-specific
antagonist, C5aRA. By using this strategy, we verified the critical
requirement of mast cells in IC-induced skin injury, while C3 plays a
more dispensable role due to compensation by C5a/C5aR triggering, which
can occur even in the absence of C3. In the lung, both mast cells and
C3 appear to contribute to the accumulation of PMN in the
bronchoalveolar space by apparently distinct mechanisms. Only the lack
of C3 correlated with reduced levels of TNF- and PMN chemotactic
cytokines, while remaining unchanged in mast cell-deficient
KitW/KitW-v mice. Together,
our data indicate for distinct tissue site-specific requirements of
complement and mast cells in IC inflammation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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C3-deficient mice were generated by targeted gene disruption, as described previously (32). These C3-/- mice and their wild-type (WT) littermates were used on a mixed 129 and C57BL/6 genetic background. Mast cell-deficient WBB6F1-KitW/KitW-v mice together with their congenic normal (WBB6F1+/+) mice were purchased from The Jackson Laboratory (Bar Harbor, ME). All these mice were male and were used at 8–12 wk of age. Experiments were conducted in accordance to the regulations of the local authorities.
Passive reverse Arthus reaction in skin and lung
The Arthus reaction in skin and lung was induced as reported
previously (25). Unless stated otherwise, all materials
were purchased from Sigma (Munich, Germany). Briefly, 30 µg rabbit
anti-OVA IgG Ab was injected intradermally at multiple sites,
followed by control injection of PBS at contralateral sites. In
addition, 150 µg Ab was applied intratracheally. Immediately
thereafter, 20 mg/kg OVA Ag was given i.v. Ab control animals received
PBS instead of OVA Ag. In some experiments, mice additionally received
400 µl C5aRA at a concentration of 7.3 x
10-6 M, as described (13, 25). Mice
were killed 4 h after OVA/anti-OVA IC challenge. The skin was
harvested, and punches of the site of injection 10 mm in diameter were
weighed. Bronchoalveolar lavage was performed with 5 x 1 ml PBS
at 4°C. Total cell count of the bronchoalveolar lavage fluid (BALF)
was assessed with a hemocytometer (Neubauer Zählkammer, Gehrden,
Germany). The amount of erythrocytes represented the degree of
hemorrhage. For quantitation of alveolar PMN accumulation, differential
cell counts were performed on cytospins (10 min at 55 x
g) stained with May-Grünwald-Giemsa using 300 µl
BALF. Plasma exudation of skin reaction was assessed by weighing skin
punches of IC reaction per mouse, subtracting the weight of sham
control specimens from contralateral PBS injection sites of the same
animal. Myeloperoxidase (MPO) activity of skin punches and lavaged lung
tissue was assayed as previously described (25). In brief,
homogenized tissue was suspended in 50 mM potassium phosphate buffer,
pH 6, 0.5% hexadecyltrimethyl ammonium bromide, subsequently exposed
to three freeze-thaw cycles, and finally sonicated. A total of 0.167
mg/ml o-dianisidine dihydrochloride and 0.0005% hydrogen
peroxide was added to the supernatant. The change in OD at 
=
450 nm was recorded. A serial dilution of MPO from human PMN
(Calbiochem-Novabiochem, Bad Soden, Germany) served as a standard.
Samples were run in duplicate.
Determination of cytokine levels in BALF
The concentrations of TNF-, cytokine-induced neutrophil
chemoattractant (KC), and macrophage-inflammatory protein (MIP)-2 in
BALF were assayed in duplicate in appropriately diluted samples with
respective TNF--, KC-, and MIP-2-specific ELISA kits (R&D Systems,
Wiesbaden, Germany), according to the manufacturer’s instructions.
Statistical analysis
Statistical analysis was performed using the SPSS V. 9.0 statistical package (SPSS, Chicago, IL). To analyze differences of mean values between groups, a two-sided unpaired Student t test was used.
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Results and Discussion |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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Recently, we had shown that the Arthus reaction, if induced by
OVA/anti-OVA IgG IC, requires both FcRIII and C5aR
(25). As summarized in Table I
, mice with either genetic deletion of FcRIII or blockade of C5aR
exhibited a substantial attenuation of the inflammatory response, with
PMN infiltration and plasma exudation being in the order 25 to 40% of
WT reaction, while a combined dysfunction of FcRIII and C5aR
virtually abrogated inflammation (25). Since IC in the
perivascular tissue are likely to encounter FcRIII-positive mast
cells positioned in the vicinity of blood vessels, we induced cutaneous
Arthus reaction in mast cell-deficient
WBB6F1-KitW/KitW-v
mice together with their Kit+/+ controls.
As shown in Fig. 1, both PMN infiltration (A) and plasma exudation
(B) were strongly attenuated in the skin of
KitW/KitW-v mice. The degree
of attenuation was similar to FcRIII-/- mice
if treated with C5aRA (Table I). These results confirm previous
findings, which suggested a major contribution of both FcRIII and
C5aR together with mast cells in immune vasculitis and cutaneous Arthus
reaction (21, 24, 25, 30).
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). However, cutaneous
deposition of OVA/anti-OVA IgG IC resulted in a profound PMN
accumulation that was indistinguishable between
C3-/- mice and WT controls, while plasma
exudation was even more pronounced in C3 deficiency (Fig. 2). Similar results had recently been obtained by others, and it was
concluded that complement plays no role in IC inflammation in general
(reviewed by Ref. 33). However, local production of C5 and
cleavage to C5a by proteases other than C5 convertase might still occur
in C3-/- mice, thus representing a potential
bypass to the classical complement cascade. In support, a substantial,
although not complete, attenuation of MPO activity and plasma exudation
was found in C3-/- mice after C5aR blockade
(Table II). C3b-independent activation of C5 in the generation of C5a may
therefore account in part for the strong inflammatory response of the
cutaneous Arthus reaction seen in C3 deficiency.
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The contribution of complement and mast cells to the Arthus
reaction may differ between tissue sites, as recently suggested by
varying degrees of attenuation of IC reactions, either in skin and
peritoneum of KitW/KitW-v
mice, or in skin and lung of mice following inhibition of C5aR
(25, 34). Similar to the situation in skin, the profound
pulmonary inflammation was indistinguishable between
C3-/- mice and WT controls, as assessed by MPO
activity and alveolar hemorrhage (Fig. 3, A and B). Interestingly, differences in the
cellular composition of skin and lung indicated for different roles of
mast cells. In the lung, mast cells are virtually absent in the
alveoli, where IC are supposed to aggregate. In contrast to skin, mast
cell-deficient KitW/KitW-v
mice showed very strong signs of pulmonary IC inflammation, with no
reduction of interstitial PMN infiltration and vascular permeability,
as compared with their mast cell-sufficient
Kit+/+ counterparts (Fig. 3, C
and D).
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The lung is known to hold a considerable pool of marginated PMN
exceeding the pool present in the circulating blood (35).
Assessment of MPO activity in homogenized lung tissues may therefore
not differentiate between intravascular PMN present in an unstimulated
situation and PMN that infiltrated into the lung tissue upon IC
stimulation. However, if alveolar inflammation induces a chemotactic
gradient, PMN would reach the alveoli, where PMN are normally not
present. We therefore examined the degree of alveolar PMN influx in
C3-/- mice and
KitW/KitW-v mice, as obtained
by bronchoalveolar lavage. At 4 h of IC challenge, BALF of control
mice contained strongly increased numbers of 355 ± 90.3 x
103 (n = 18) of alveolar PMN in
C3+/+ WT mice, and of 396.7 ± 126.4 x
103 (n = 10) in mast
cell-sufficient Kit+/+ mice. These
neutrophil numbers in BALF were largely reduced in
C3-/- mice, or
KitW/KitW-v mice (Fig. 4).
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, MIP-2, and KC suspected to be involved in the
recruitment of neutrophils. While TNF- induces adhesion molecules,
thus promoting PMN migration more indirectly, the two ELR-CXC
chemokines MIP-2 and KC have direct chemotactic properties for PMN
(36, 37, 38). As shown in Fig. 5, C3 deficiency led to a significant decrease of all mediators
investigated. In addition, chemotactic activity within BALF of
C3-/- mice showed reduction of 
40% of PMN
migration in vitro (data not shown). In sharp contrast, cytokine levels
remained largely unchanged in mast cell deficiency (Fig. 5).
Differences in the cytokine production of Kit+/+
and C3+/+ control mice were also evident and may
be due to the distinct genetic background of WBB6 in
Kit+/+ mice vs 129B6 in
C3+/+ mice. Kit+/+ mice
produce substantially lower amounts of cytokines upon IC challenge than
do C57BL/6 and 129 mice (data not shown), and 129B6
C3+/+ mice (Fig. 5), while not further reduced in
KitW/KitW-v mice (Fig. 5).
These findings argue for different pathways in IC-induced lung injury
by which complement component C3 and mast cells trigger recruitment
of PMN.
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, residual
inflammation still occurs in C3-/- mice after
C5aR inhibition, and this appears to be determined by
FcRIII-triggered activation of mast cells (15, 21). The
phenotypes obtained with FcRIII-/- mice,
C5aR-antagonized FcRIII-/- mice, and
KitW/KitW-v mice are in
accordance with the view that C5aR and FcRIII (both expressed by
mast cells) represent the major receptor systems in the initiation of
the skin Arthus reaction (25).
An interesting observation is that mast cells, as well as C3,
contribute to some acute changes associated with the lung Arthus
reaction, especially alveolar PMN migration, while interstitial PMN
accumulation and hemorrhage both appear C3 and mast cell independent.
The alveolar macrophage is known as a major effector cell promoting the
recruitment and activation of neutrophils through the production of
cytokines such as TNF- and CXC chemokines like MIP-2 and KC upon
triggering FcRIII and C5aR (16, 29, 40 ;
unpublished observations). These mediators are also reduced in C3
deficiency, indicating for a common pathway in the activation of
macrophages by C3, or C5aR, or FcRIII, all contributing to enhanced
synthesis of TNF- and chemokines and subsequent PMN transmigration
from lung tissue into alveoli (40). The experiments with
KitW/KitW-v mice further show
that mast cells can play an additional role. Surprisingly, the mast
cell-dependent in vivo cytokine profile is different from that
described for macrophages, and the production of TNF-, MIP-2, and KC
remained completely unaffected by mast cell deficiency in pulmonary IC
inflammation. This may suggest that the contribution of mast cells to
intraalveolar accumulation of neutrophils occurs by the release
of effector molecules other than TNF- or CXC chemokines. Among
several potential candidates, the relative role of leukotriene
B4 and platelet-activating factor is currently
under investigation (41).
In summary, we have used
KitW/KitW-v- and C3-deficient
mice, the latter in combination with a specific antagonist against
C5aR, to distinguish among mast cell-, C3-, and C5aR-mediated effects.
This approach enabled us to demonstrate that complement and mast cells
have distinct tissue site-specific requirements acting by apparently
different mechanisms in the initiation of IC disease. Our findings
support the current model that mast cells are the dominant effector
cell type in skin (21, 24). In the lung, mast cells can
also contribute, but other effector cells (alveolar macrophages,
endothelial cells, etc.) are certainly of additional importance for
triggering the full inflammatory response (29).
Furthermore, we provided evidence that, in response to IgG IC formation
in skin, the C5aR-mediated receptor pathway is operative in
C3-/- mice, and therefore not entirely
dependent of C3 synthesis. This provides an explanation why
C3-/- and C5aR-/- mice
develop such different phenotypes in IC inflammation, previously
considered as contradictory data (for review, see Refs. 33, 42). Finally, C3 (similar as FcRIII and C5aR) (25, 40 , and data not shown) was found to contribute to the
elicitation of PMN to alveoli that correlated with increased synthesis
of TNF- and CXC chemokines. In this respect, IC lung injury is
dependent on all C3-, C5aR-, and FcRIII-triggered activation of mast
cells/macrophages, thus strengthening the view that both complement and
FcRIII are necessary to initiate the full expression of the
inflammatory cascade.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. J. Engelbert Gessner, Abteilung für Klinische Immunologie, Medizinische Hochschule Hannover, 30625 Hannover, Germany. E-mail address: Gessner.Johannes{at}MH-Hannover.de
3 Abbreviations used in this paper: IC, immune complex; BALF, bronchoalveolar lavage fluid; C5aRA, antagonist for C5aR; MIP, macrophage-inflammatory protein; MPO, myeloperoxidase; PMN, polymorphonuclear leukocyte; WT, wild type; KC, cytokine-induced neutrophil chemoattractant.
Received for publication March 13, 2001. Accepted for publication May 10, 2001.
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. J. Immunol. 162:2321.RIII (CD16) deficient mice. Immunity 5:181.[Medline]
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RIII-mediated production of TNF induces immune complex-alveolitis independent of CXC chemokine generation. J. Immunol. 166:5193.-receptors in the Arthus reaction. Mol. Immunol. 36:893.[Medline]This article has been cited by other articles:
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 A. Braun, J. E. Gessner, D. Varga-Szabo, S. N. Syed, S. Konrad, D. Stegner, T. Vogtle, R. E. Schmidt, and B. Nieswandt STIM1 is essential for Fc{gamma} receptor activation and autoimmune inflammation Blood, January 29, 2009; 113(5): 1097 - 1104. [Abstract] [Full Text] [PDF]
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