A Codominant Role of FcγRI/III and C5aR in
the Reverse Arthus Reaction
This information is current as
of June 17, 2017.
Ulrich Baumann, Jörg Köhl, Thomas Tschernig, Kirsten
Schwerter-Strumpf, J. Sjef Verbeek, Reinhold E. Schmidt
and J. Engelbert Gessner
J Immunol 2000; 164:1065-1070; ;
doi: 10.4049/jimmunol.164.2.1065
http://www.jimmunol.org/content/164/2/1065
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References
A Codominant Role of Fc␥RI/III and C5aR in the Reverse
Arthus Reaction1
Ulrich Baumann,* Jörg Köhl,† Thomas Tschernig,‡ Kirsten Schwerter-Strumpf,*
J. Sjef Verbeek,§ Reinhold E. Schmidt,* and J. Engelbert Gessner2*
I
mmunoglobulin G-containing immune complexes (IC)3 contribute to the pathophysiology in a number of autoimmune
diseases, exemplified by systemic lupus erythematosus, rheumatoid arthritis, Goodpasture’s syndrome, and hypersensitivity
pneumonitis (1– 4). The classical animal model for the inflammatory response in these IC diseases is the Arthus reaction, which
features the infiltration of polymorphonuclear cells (PMN), hemorrhage, and plasma exudation (5).
It has long been accepted that IC-mediated activation of complement via the classical pathway represents the initial trigger of
the Arthus response. The ensuing inflammation is mediated by a
group of proinflammatory molecules, the lytic membrane attack
complex C5b-9 and the anaphylatoxins C3a and C5a. C5a leads to
increased vascular permeability and promotes migration and activation of leukocytes (6). It’s not only a potent chemotactic agent
for PMN, mast cells, and monocytes, it also enhances adhesion of
PMN to endothelial cells and induces mediator release from leukocytes (7, 8). Studies with C5aR-deficient mice demonstrate a
strongly reduced Arthus reaction in skin, lung, and peritoneum (9).
A similar degree of attenuation occurs after blocking C5a-C5aR
interaction with a specific C5aR antagonist (C5aRA), indicating
*Department of Clinical Immunology, †Institute of Medical Microbiology, and ‡Department of Functional Anatomy, Medical School Hannover, Hannover, Germany;
and §Department of Human and Clinical Genetics, Leiden University Medical Center,
Leiden, The Netherlands
Received for publication August 9, 1999. Accepted for publication October 26, 1999.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by a fellowship to U.B. from the HiLF Programme of
Hannover Medical School. The transgenic and other research were supported by
grants from the Deutsche Forschungsgemeinschaft to R.E.S. and J.E.G. (Ge892/5-1,
Ge892/7-1, SFB 265/B1) and by a grant of the Bundesministerium für Bildung, Wissenschaft, Forschung und Technologie to J.K. (01VM9305).
2
Address correspondence and reprint requests to Dr. J. Engelbert Gessner, Abteilung
für Klinische Immunologie, Medizinische Hochschule Hannover, 30625 Hannover,
Germany. E-mail address: [email protected]
3
Abbreviations used in this paper: IC, immune complex; BAL, bronchoalveolar lavage; C5aRA, C5aR antagonist; MPO, myeloperoxidase; PMN, polymorphonuclear
leukocytes; WT, wild type; CVF, cobra venom factor.
Copyright © 2000 by The American Association of Immunologists
that the complement effect in IC inflammation is predominantly
mediated by C5aR (10).
During the last few years, studies using mice deficient in Fc
receptors for IgG (Fc␥R) have revealed a critical role of Fc␥R,
especially Fc␥RIII, in the pathogenesis of IC diseases (11–13). It
has been proposed that the initiation of the inflammatory cascade
depends almost entirely on mast cell Fc␥R-triggered activation
leading to secondary reactions such as local complement production (14, 15). Supporting this concept, mice lacking FcR␥ (deficient in both Fc␥RI and Fc␥RIII), but not C3-, C4-, and C5-deficient mice, show an impaired Arthus reaction (16). However, not
all results are in agreement with that redefinition. First, complement-sufficient and complement-depleted Fc␥RIII-deficient mice
show that IC-mediated cutaneous Arthus reaction is induced via a
complement-dependent and a Fc␥RIII-dependent pathway (17). Second, it is unclear why C5-deficient mice show no impaired cutaneous
inflammation, whereas a marked reduction has been described for
other organs, including lung and peritoneum (16, 18, 19). Third, the
contribution of complement and/or Fc␥R may differ with the genetic
background. This has been demonstrated for PMN recruitment in an
IC peritonitis model, which is more strongly affected by complementdepletion in BALB/c mice compared with C57BL/6 mice (20).
In the present study, we examined the responses to IC activation
by combining several Fc␥R deficiencies with dysfunction of complement, specifically at the level of the C5aR. We secured comparability of the different mouse strains by using wild-type (WT)
and Fc␥RIII- and FcR␥-deficient mice all on the same genetic
background. To study the Arthus reaction comprehensively, we
simultaneously assessed the immunopathology in distinct organs,
i.e., skin and lung, of the same animal, employing several parameters for each. By using this strategy, we identified dependent and
independent contributions of both Fc␥RI/III- and C5aR-triggered
pathways, which gives an explanation of why these two effector
systems previously have been considered to dominate each other.
Materials and Methods
Mice
Fc␥RIII-deficient mice were generated as decribed (17). They were bred
for eight generations onto C57BL/6 mice under pathogen-free conditions in
the animal facility of Hannover Medical School. The homozygous
0022-1767/00/$02.00
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Recent attempts to specify the relative contribution of FcR and complement in various experimental systems of immune complex
disease have led to opposing conclusions. As concluded in IgG FcR␥ⴚ/ⴚ mice, manifestation of disease is almost exclusively
determined by Fc␥R on effector cells, arguing for a minor role of complement. In contrast, data obtained with C5aRⴚ/ⴚ mice
suggested that, dependent on the tissue site, complement is more important than Fc␥R. In this paper, we demonstrate that, in
response to IgG immune complex formation, Fc␥RI/III- and C5aR-mediated pathways are both necessary and only together are
they sufficient to trigger the full expression of inflammation in skin and lung. Moreover, both effector systems are not entirely
independent, suggesting an interaction between Fc␥R and C5aR. Therefore, Fc␥R-mediated responses can be integrated through
C5aR activation, which may explain why these two receptor pathways have previously been considered to dominate each
other. The Journal of Immunology, 2000, 164: 1065–1070.
1066
Fc␥RIII⫺/⫺ mice were selected and WT Fc␥RIII⫹/⫹ C57BL/6 littermates
were used for all comparisons. C57BL/6 mice homozygous for FcR␥⫺/⫺
were obtained from Taconic (Germantown, NY). All these mice were male
and were used at 8 –12 wk of age. Experiments were conducted in accordance with the regulations of the local authorities.
Reverse passive Arthus reaction in skin and lung
Mice were anesthetized with ketamine and xylazine and shaved at their
basolateral sides. Rabbit IgG anti-OVA Ab (30 ␮g; Sigma, Munich, Germany) was injected intradermally at multiple sites. In addition, the trachea
was cannulated and 150 ␮g Ab was applied. Immediately thereafter, 200 ␮l
of 0.25% Evans blue together with 20 mg/kg of OVA Ag were given i.v.
Where indicated, mice were injected twice with 4.25 ␮g purified cobra
venom factor (CVF) (Naja naja, Calbiochem-Novabiochem, Bad Soden,
Germany) i.p. at 24 and 16 h before the Arthus reaction to deplete complement. Serum complement levels were determined as described (21). In
additional experiments, mice received C5aRA ⌬pIIIA8 to inhibit C5aRtriggered activation (10). Hereby, 200 ␮l antagonist was given at a concentration of 7.3 ⫻ 10⫺6 M before application of anti-OVA IgG, and then
100 ␮l antagonist was given at 60 and 120 min after IC challenge. Mice
were killed 4 h after initiation of the Arthus reaction and were assayed for
infiltration of PMN, hemorrhage, and plasma exudation in skin, lavaged
lung tissue, and bronchoalveolar lavage (BAL) fluid.
Pulmonary vasculature was gently flushed with PBS with a catheter positioned in the root pulmonary artery. Lungs were lavaged with PBS (1 ml,
five times at 4°C) after cannulation of the trachea. The volume of collected
BAL fluid was measured in each sample and total cell count was assessed
with a hemocytometer (Neubauer Zählkammer, Gehrden, Germany). The
amount of erythrocytes represented the degree of hemorrhage. For quantitation of PMN accumulation, differential cell counts were performed on
cytospins (10 min at 55 ⫻ g) stained with May-Grünwald/Giemsa using
300 ␮l of BAL fluids.
favorably with the methodology involving radiolabeled albumin (23). Onecentimeter skin punches were harvested, placed in 1 ml of formamide,
sonicated for 10 min, and incubated at 37°C for 16 h. The concentration of
dye in appropriate dilutions of skin eluate, BAL fluid, and serum was
measured spectrophotometrically at 620 nm. Serum samples were also
measured at 405 nm to account for hemolysis. Extinction at 405 nm was
multiplied with factor 0.014 and deducted from the extinction at 620 nm.
This factor corrects for extinction caused by murine hemoglobin at 620 nm.
Plasma exudation was quantitated by multiplying the ratio of extinction in
BAL fluid or skin punches to extinction in plasma by the dilutions of the
specimens.
Histologic and immunohistochemistry studies
To process lung tissues for histological examination after lavage, they were
fixated in 4% buffered paraformaldehyde, embedded in paraffin, and
stained with hematoxylin and eosin according to conventional procedures.
In additional experiments, lavaged lung tissues were prepared for immunohistochemistry. The alkaline phosphatase antialkaline phosphatase
(APAAP) technique was used to phenotype neutrophils (Gr1; Dianova,
Hamburg, Germany). Cryostat sections were incubated with the primary
Abs for 30 min at 21°C. After washing with TBS-Tween (0.05% Tween
20; Serva, Heidelberg, Germany), incubations were done with the bridging
Ab Z0494 and the rat-APAAP Ab complex D0488 for 30 min. To increase
the staining intensity, the last two steps were repeated once. Fast blue
(Sigma) served as substrate for alkaline phosphatase. Positive and negative
controls produced the expected results.
Statistical analysis
Statistical analysis was performed using the SPSS v. 8.0 statistical package
(SPSS, Chicago, IL). To analyze differences of mean values between
groups, a two-sided unpaired Student‘s t test was used; p ⬍ 0.05 was
considered significant and p ⬍ 0.001 was considered highly significant.
Independent contribution of Fc␥R and C5aR was assessed by two-way,
two-factorial ANOVA (univariate general linear model procedure). Function or dysfunction of either receptor was coded in a binary variable (factor). Between-subject effects indicated independent contribution of a factor.
Myeloperoxidase (MPO) assay in skin and lung
Skin punches of the injection sites 1 cm in diameter, lavaged lung tissues,
and BAL fluids were assessed for PMN accumulation by MPO activity as
described (22). Briefly, homogenized tissue was suspended in 50 mM potassium phosphate buffer (pH 6.0, 0.5% hexadecyltrimethylammoniumbromide). Cells were broken by three cycles of freezing and thawing and
subsequent sonication. The supernatant was mixed with 0.167 mg/ml odianisidine dihydrochloride (Sigma) and 0.0005% hydrogen peroxide. The
amount of MPO was calculated by assessing the change in absorbance at
450 nm. A serial dilution of MPO from human PMN (Calbiochem-Novabiochem) served as standard. Samples were run in duplicate.
Measurement of plasma exudation in skin and lung
Evans blue dye binds avidly to albumin and serves as a marker of extravasation of plasma proteins into skin and lung. This technology compares
Results and Discussion
Arthus reaction in the skin
Reverse passive Arthus reactions in the skin were performed to
dissect the relative contribution of Fc␥RI, Fc␥RIII, and complement during cutaneous, IC-triggered inflammation. C57BL/6 mice
deficient in the ligand-binding ␣-chain of Fc␥RIII (Fc␥RIII⫺/⫺) or
deficient in the FcR␥-chain critical for signaling of both Fc␥RIII
and Fc␥RI (FcR␥⫺/⫺) were compared with WT C57BL/6 mice.
Inflammatory responses were quantitated by measurement of MPO
in homogenized tissue as a marker of PMN infiltration and by
assessment of Evans blue in tissue extracted by formamide, indicating the degree of microvascular permeability. In WT mice, a
FIGURE 1. Passive reverse cutaneous Arthus reaction in WT, Fc␥RIII⫺/⫺, and FcR␥⫺/⫺ mice receiving CVF and C5aRA. Mice were injected intracutaneously with 30 ␮g of IgG anti-OVA Ab and then with systemic 20 mg/kg OVA Ag and 0.25% Evans blue. Recruitment of PMN (A) and plasma
exudation (B) were assessed 4 h after initiation of the cutaneous Arthus reaction by MPO assay or formamide extraction of extravasated blue dye,
respectively (IC, f). Treatments with CVF and C5aRA are indicated (IC ⫹ CVF, u; and IC ⫹ C5aRA, ^). Mice receiving only Ag or Ab served as controls
(Ag, 䡺; and Ab, dotted bars). Ag control group and Ab control group each comprised five WT mice per group, and IC treatment groups comprised eight
to twelve mice per group (except for IC ⫹ C5aRA, which had five to six mice per group). Data are expressed as mean ⫾ SEM. Differences for both
parameters were significant or highly significant for the IC treatment groups of WT mice compared with Fc␥RIII⫺/⫺ and FcR␥⫺/⫺ mice (ⴱ, p ⬍ 0.05 to
p ⬍ 0.001), and were significant for IC compared with IC ⫹ CVF and IC ⫹ C5aRA treatment groups (†, p ⬍ 0.05). In addition, Fc␥RIII⫺/⫺ and FcR␥⫺/⫺
mice differed significantly for plasma exudation but not PMN infiltration (‡, p ⬍ 0.05).
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BAL and quantitation of hemorrhage and PMN accumulation in
bronchoalveolar space
Fc␥R AND C5aR IN IMMUNE COMPLEX DISEASE
The Journal of Immunology
1067
Arthus reaction in the lung
It is still unclear whether the pathogenesis of the inflammatory
response in IC diseases follows a general pattern (13) or if tissuespecific differences exist (9). Thus, the immunopathological cascade was induced not only in the skin, but also simultaneously in
the lung in the same animals, allowing for an assessment of organspecific differences under exactly the same experimental conditions. In WT mice, the profound pulmonary IC inflammatory response with enhanced PMN infiltration, plasma exudation, and
hemorrhage into the bronchoalveolar space was markedly suppressed, although not completely abolished, by application of either CVF or C5aRA (Fig. 2A, C, and D). Interestingly, interstitial
PMN influx, as defined by MPO activity in lavaged lung tissue,
was substantially attenuated after C5aRA treatment, whereas systemic complememt depletion by CVF resulted in increased MPO
activity (Fig. 2B). This finding is in accordance with the recognized ability of CVF to induce accumulation of PMN, especially in
the lung (25).
Given the primary role of C5a among complement mediators in
both skin and lung, the contribution of Fc␥RI and Fc␥RIII to the
pathophysiology in the lung was further evaluated. In both
Fc␥RIII⫺/⫺ and FcR␥⫺/⫺ mice, alveolar PMN infiltration as well
as interstitial PMN accumulation in lavaged lung tissue was substantially decreased (Fig. 3, A and B). The reduction in hemorrhage
FIGURE 2. Pulmonary IC inflammation in WT mice after CVF and
C5aRA treatment. The induction of the inflammatory response in the lung
was performed by challenge with 150 ␮g anti-OVA Ab intratracheally and
then with OVA Ag in WT mice (IC, f), WT mice treated with CVF (IC
⫹ CVF, u), and C5aRA (IC ⫹ C5aRA, ^). Mice receiving only Ag or Ab
served as controls (Ag, 䡺; and Ab, dotted bars). After 4 h, mice were killed
and PMN infiltration in the alveolar space (A), assessed by differential cell
counts in Giemsa stains of BAL fluid, PMN infiltration in lung tissue (B),
measured by MPO activity in lavaged lung, hemorrhage (C), measured by
total cell count of erythrocytes present in BAL fluid, and plasma exudation
(D), quantitated by the amount of Evans blue dye extravasation in BAL
fluid, were evaluated. Ag and Ab control groups comprised 5 animals per
group, and IC treatment groups comprised 8 –12 animals per group (except
for C5aRA, which had five to six animals per group). Data are presented as
mean ⫾ SEM. Differences in IC compared with IC ⫹ CVF and IC ⫹
C5aRA treatment groups were significant or highly significant for all parameters (ⴱ, p ⬍ 0.05 to p ⬍ 0.001).
seen in Fc␥RIII⫺/⫺ mice was further attenuated in FcR␥⫺/⫺ mice
(Fig. 3C). This result shows that both Fc␥RI and Fc␥RIII contribute significantly to this parameter. Unexpectedly, plasma exudation in the lung was largely independent of Fc␥RI and Fc␥RIII
(Fig. 3D), which is different than what has been observed in the
skin (Fig. 1B). This finding was confirmed by histological studies
demonstrating marked perivascular edemas in WT mice (Fig. 4A)
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strong accumulation of PMN with a concomitant increase of
plasma exudation was observed within 4 h after IC challenge (Fig.
1, A and B). In Fc␥RIII⫺/⫺ mice, recruitment of PMN was markedly decreased and comparable to that seen in FcR␥⫺/⫺ mice (Fig.
1A). Cutaneous plasma exudation differed in that the attenuation
observed in Fc␥RIII⫺/⫺ was further reduced to background levels
in FcR␥⫺/⫺ mice (Fig. 1B). These data identify Fc␥RIII as the
dominant FcR in PMN influx and suggest that both Fc␥RI and
Fc␥RIII can trigger enhanced vascular permeability.
Recently, we verified the proposed role of complement (24) in
addition to Fc␥R in cutaneous Arthus reaction. This was based on
the observation that complement depletion with CVF completely
abrogated the inflammatory response, as defined by Evans blue
extravasation, only in Fc␥RIII⫺/⫺ mice and not in control mice
(17). The Fc␥RIII⫺/⫺ mice and their normal littermates used in
that study had a mixed genetic background of C57BL/6 and 129
strains. Interestingly, although rather ineffective in mixed
C57BL/6 and 129 strains, the treatment with CVF resulted in a
significant attenuation of inflammation in WT mice of C57BL/6
strain. By using two i.p. injections of 4.25 ␮g CVF 24 and 16 h
before IC challenge, a strong reduction of PMN influx and a complete suppression of plasma exudation were observed (Fig. 1, A
and B). This finding illustrates that variations in the genetic background of C57BL/6 and 129 mice are of strong influence of the
inflammatory response to the Arthus reaction. In support, the hemolytic activity is significantly lower in C57BL/6 mice compared
with that in 129 mice (17), which may indicate that strain-specific
differences in complement activity can contribute to the observed
heterogeneity. The importance of complement was further confirmed with the specific C5aRA ⌬pIIIA8 (10). In WT mice, MPO
activity and plasma exudation decreased by more than 60 and 80%,
respectively, after C5aRA treatment (Fig. 1, A and B). This indicates a critical role for C5a, as recently suggested in C5aR⫺/⫺
mice (9). A complete suppression for both parameters was similarly observed in Fc␥RIII⫺/⫺ mice and in FcR␥⫺/⫺ mice after
inhibition of C5aR-triggered activation by C5aRA (Fig. 1, A and
B). Taken together, these results demonstrate that Fc␥R and C5aR
are codominant receptor pathways in the initiation of the inflammatory cascade in skin of C57BL/6 mice.
1068
which were also evident in Fc␥RIII⫺/⫺ mice (Fig. 4B) and
FcR␥⫺/⫺ mice (Fig. 4C). The residual proportion of hemorrhage
and plasma exudation seen in Fc␥RIII-deficient mice was completely abrogated after C5aRA treatment (Fig. 3, C and D). In
contrast, interstitial MPO activity of Fc␥RIII⫺/⫺ mice and
FcR␥⫺/⫺ mice was not affected, and alveolar PMN influx was
totally absent after inhibition of C5aR (Fig. 3, A and B). As shown
by immunohistochemistry, Gr1-positive PMN in C5aRA-treated
FcR␥⫺/⫺ mice retarded in the pulmonary vasculature, apparently
unable to migrate along a chemotactic gradient into the alveolar
space (Fig. 4D).
Together, the results give strong evidence that in C57BL/6 mice
Fc␥R and C5aR are codominant receptors in the initiation of the
Arthus response in both skin and lung and further suggest that the
sequelae of IC-triggered inflammation are not necessarily the endpoints of the same reaction. Tissue site-specific differences exist
with respect to plasma exudation, which is almost entirely dependent on C5aR in the lung and on both C5aR and Fc␥R in the skin.
Fc␥RIII is the dominant Fc␥R for cutaneous and alveolar PMN
influx. Interestingly, Fc␥RI is of additional importance for plasma
exudation in the skin and hemorrhage in the lung. These observations, combined with the recent report that Fc␥RI contributes significantly to enhanced PMN infiltration in IC-induced peritonitis
(20), indicate that effector cells expressing Fc␥RI are involved in
IC-triggered inflammation. In the lung, the alveolar macrophage
expressing Fc␥RI, Fc␥RII, and Fc␥RIII is the most prominent cell
type in the alveoli and is known to promote IC injury through the
production of cytokines (TNF-␣, platelet-activating factor, etc.),
chemokines (macrophage-inflammatory protein-2, cytokine-induced neutrophil chemoattractant-1, etc.), and vasoactive substances (reviewed in Ref. 26). In the skin, resident mast cells,
epidermal Langerhans cells (both of which express Fc␥RIII), and
macrophages are the most likely involved cell types (15, 27).
These mast cells have already been demonstrated to contribute
significantly to IC-induced injury in skin (15, 28). However, because Fc␥RIII, but not Fc␥RI, is expressed on mast cells, it appears
that skin macrophages are of additional importance.
One might assume that Fc␥R and complement represent a redundant system, leading to less attenuation by dysfunction of one
pathway with the remaining pathway partly substituting the response. However, as shown in this study, the sum of attenuations
caused by Fc␥R deletion and C5aR blockade is far above 100% in
all parameters analyzed. Therefore, both pathways may in part act
independently, but also have to act in a cascade of events with one
pathway dependent on triggering by the other as first suggested by
Sylvestre and Ravetch (14). Action independent from the other
pathway can be derived from the inflammatory response present
with one pathway functional and the other dysfunctional. This has
been formally assessed by ANOVA ( p values for between-subject
effects of the factors Fc␥R and C5aR, respectively: alveolar PMN
infiltration, 0.007, 0.002; pulmonary interstitial PMN infiltration,
0.026, 0.307; alveolar hemorrhage, 0.003, 0.058; pulmonary
plasma exudation, 0.196, 0.002; cutaneaous PMN infiltration,
0.007, 0.002; and cutaneous plasma exudation, 0.017, 0.15). This
demonstrates that the Fc␥R and C5aR pathways contribute significantly to the inflammatory response, with the exceptions of C5aR
in interstitial PMN influx and Fc␥R in pulmonary plasma exudation. On the other hand, the sum of the independent contributions
obtained by adding the mean values of single groups does not
reach the full strength of the inflammatory response as seen in WT
mice. This fact suggests a dependent interaction between Fc␥R and
C5aR which may explain the controversy about their relative roles
(9, 13).
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FIGURE 3. Pulmonary IC inflammation in WT, Fc␥RIII⫺/⫺, and
FcR␥⫺/⫺ mice receiving C5aRA. Inflammation in the indicated mice (IC,
f) was induced and analyzed for PMN infiltration in alveoli (A), MPO
activity in lavaged lung tissue (B), alveolar hemorrhage (C), and alveolar
plasma exudation (D) as described in Fig. 2. Where indicated, mice were
additionally treated with 3⫻ C5aRA in a total volume of 400 ␮l at a
concentration of 7.3 ⫻ 10⫺6 M (IC ⫹ C5aRA, ^). WT mice receiving PBS
instead of Ag (not shown) or Ab served as controls (Ab, dotted bars). Ab
control groups comprised 5 animals per group, IC treatment groups comprised 8 –12 animals per group, and C5aRA-treated mice comprised five to
six animals per group. Data are presented as mean ⫾ SEM. Differences for
hemorrhage, and alveolar and interstitial PMN infiltration were significant
for the IC treatment groups of WT mice compared with Fc␥RIII⫺/⫺, and
FcR␥⫺/⫺ mice (ⴱ, p ⬍ 0.05), whereas the decrease in plasma exudation
was not significant with p ⫽ 0.274 and p ⫽ 0.097, respectively.
Fc␥RIII⫺/⫺ and FcR␥⫺/⫺ mice only differed significantly for hemorrhage
(‡, p ⬍ 0.05). Animals treated with C5aRA differed significantly compared
with untreated mice of the same genotype in alveolar PMN infiltration,
alveolar plasma exudation, and pulmonary hemorrhage (†, p ⬍ 0.05) with
the exception of hemorrhage in FcR␥⫺/⫺ mice (p ⫽ 0.202). No differences
were observed for MPO activity in lavaged lung tissue. As in the skin, Ag
and Ab control values did not differ between WT and FcR⫺/⫺ mice (data
not shown).
Fc␥R AND C5aR IN IMMUNE COMPLEX DISEASE
The Journal of Immunology
1069
In summary, we have used Fc␥RIII- and FcR␥-deficient mice in
combination with a specific antagonist against C5aR to distinguish
between Fc␥RI-, Fc␥RIII-, and C5aR-mediated effects. This approach enabled us to demonstrate that the initiation of the inflammatory cascade in IC disease is mainly determined through the
action of Fc␥RIII and C5aR pathways. Furthermore, the comparison between Fc␥RIII⫺/⫺ and FcR␥⫺/⫺ mice shows that Fc␥RI
plays an additional role. The earlier observations of C5aR⫺/⫺ mice
(9) suggested a strict tissue-site dependency of C5aR. We observed
minor differences for both Fc␥R and C5aR, with the exception that
vascular permeability was more prominently mediated by C5aR in
lung and by Fc␥R in skin. In addition, our findings not only support the current idea that Fc␥RIII is a dominant receptor in acute
inflammation (17) but also extend this concept by integrating the
interplay between Fc␥RI, Fc␥RIII, and C5aR with dependent and
independent actions. Finally, the data strengthen the view that both
Fc␥R and C5aR have to be considered as potential targets in immunotherapy of IC disease in humans.
Acknowledgments
We thank the members of our laboratory and H. Hecker (Department of
Biomedical Statistics) for valuable discussions on the manuscript.
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FIGURE 4. Histopathology of lung from Fc␥R-deficient mice. A–C, Representative hematoxylin and eosin-stained sections of lungs from WT (A),
Fc␥RIII⫺/⫺ (B), and FcR␥⫺/⫺ (C) mice after induction of IC inflammation as described in Fig. 2 (v, vessel; b, bronchiolus; and e, edema). Marked
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mice treated with C5aRA. GR1-positive PMN (blue) are detectable sticking at the vascular wall (v). As assessed by two blinded reviewers in three to five
mice per C5aRA treatment group, this finding could be obtained in both FcR␥⫺/⫺ (D) and Fc␥RIII⫺/⫺ (not shown) mice, but not in WT mice (not shown).
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