By
From the * Noda Institute for Scientific Research, Noda-shi, Chiba-ken 278, Japan; Section of
Pulmonary and Critical Care Medicine, Department of Internal Medicine, Yale University School of
Medicine, New Haven, Connecticut 06520-8013; § Alexion Pharmaceuticals Inc., New Haven,
Connecticut 06511;
Department of Veterinary Surgery, College of Agriculture, Osaka Prefecture
University, Sakai, Osaka 593, Japan; and the ¶ Section of Allergy and Clinical Immunology,
Department of Internal Medicine, Yale University School of Medicine, New Haven, Connecticut
06520-0813
Complement (C) is an important component of innate immunity, and was also shown recently to participate in induction of acquired B cell humoral immunity. In this study, we present evidence that C also participates in acquired T cell immunity.
We found that C was involved in early events of the efferent elicitation phase of contact sensitivity (CS), and delayed-type hypersensitivity (DTH). Thus, CS and DTH were inhibited by
administration of a C-blocker, soluble recombinant C receptor-1 (sCR1), when given 30 min
before, but not 3 h after local antigen challenge. Among C components, local C5 were thought
crucial to elicitation of CS, since local administration of anti-C5 monoclonal antibodies or locally injected C-depleting cobra venom factor also inhibited CS and DTH. These findings
were consistent with our previous finding of the importance of C5 for CS elicitation, using
congenitally C5-deficient mice. To dissect the mechanism of C dependence in CS, we demonstrated that locally increased early macrophage chemotactic activity (probably C5a) in evolving
CS skin extracts, as well as late elaboration of IFN-, were both inhibited by anti-C treatment. In addition, histological analysis showed that leukocyte recruitment into CS ear sites was similarly C-dependent. Furthermore, an initiating role of B cell-derived C-fixing immunoglobulin
was suggested by demonstration of impaired CS responses in B cell-deficient mice.
In summary, these results suggest that C was activated locally, perhaps via a B cell product, in
an important early component of the stepwise events necessary to elicit CS, leading to local production of C5-dependent macrophage chemotactic activity and later IFN-, and subsequently leading to cell infiltration, for development of T cell-dependent CS.
Complement (C) is a major component of innate immunity, and is involved in early protective immune
responses against pathogens, which occur before induction
of acquired T and B cell immunity (1). Furthermore, recent findings demonstrate that innate immunity interacts
with acquired immunity (1); for example, innate immunity directs Th-1 versus Th-2 development via IFN- CS is a classical example of a T cell-mediated cutaneous
inflammatory response (13). CS and related DTH are mediated generally by Ag/MHC class II-restricted Th-1 cells,
which are recruited in mice to the local tissue site via serotonin (5-HT)-mediated processes which occur early after
Ag challenge (14). Thus, local Ag challenge causes an early
2-h release of 5-HT from tissue mast cells (14) and platelets
(15, 16), leading to endothelial cell activation via their
5-HT receptors. This enables circulating Th-1 cells to extravasate into the local site of Ag challenge, after this early
initiating phase of CS and DTH, to constitute the classical
24-h tissue swelling response. Released 5-HT also may costimulate recruited Th-1 cells via their surface 5-HT2 receptors (17, 18). Then, there are late events of the cascade
leading to CS elicitation, in which local APC activate the
recruited Th-1 cells to produce proinflammatory lymphokines such as IFN- In the course of screening for immunomodulators which
might specifically affect certain immune responses in vivo,
such as Ab production versus DTH (23), we found that Actinomyces produced a DTH-specific immunosuppressant which
was identified previously as a C5a antagonist (24). C5a
is a peptide fragment derived from cleavage of C5 during C
activation. C5a is known to be important in local immune
inflammation, and in elimination of microbes, via C5a
receptors on various cells, especially neutrophils, macrophages, and mast cells (28). Thus, C5a mediates chemotaxis, mast cell degranulation, vascular permeability, smooth
muscle contraction (29, 30), and possibly 5-HT release from
platelets (31). Since 5-HT release from mast cells (14) and
platelets (15, 16), was demonstrated to be important in
early events of CS, we previously suggested a role of C5
early in the initiation of CS by using congenitally C5-deficient mice (32).
In this study, we establish a role for C5 in cutaneous T
cell CS and DTH responses in normal mice, employing
C-depleting soluble recombinant C receptor-1 (sCR1)
(33), and cobra venom factor (CVF), and also anti-C5
mAb for C5 depletion (36, 37). We demonstrate that local
C5 acted in the early initiating phases of elicitation of CS to
mediate production of C-derived macrophage chemotactic
activity in CS ear extracts, and later IFN- Mice.
Specific pathogen-free female CBA/J, ICR, BDF1,
C57Bl/6, and B cell-deficient C57Bl/6-Igh-6 (µMT) mice (6-8
wk old) were obtained from the Jackson Laboratory (Bar Harbor,
ME), and were rested at least 1 wk before use.
Reagents.
Picryl chloride (PCl), obtained from Nacalai Tesque,
Inc. (Tokyo, Japan) was recrystallized twice as described previously (14), and stored protected from light. Zymosan and CVF were purchased from Sigma Chemical Co. (St. Louis, MO). SRBC and
anti-SRBC polyclonal Ab were products of Organon Teknika
(Durham, NC). Trinitrobenzene sulfate sodium salt was obtained
from Wako Chemicals (Osaka, Japan). Normal mouse serum,
drawn freshly from naive ICR mice via cardiac puncture, was
stored at CS Responses.
CBA/J mice were contact sensitized by topical
application of 100 µl of 5% PCl in absolute ethanol and acetone
(4:1) applied to the shaved chest and abdomen. 4 d later, CS was
elicited by painting both ears via topical application of 10 µl of a
low challenge dose of 0.4% PCl in acetone and olive oil (1:1),
compared to conventionally employed high dose of 0.8% PCl,
unless otherwise noted, since an effect of C alteration on CS usually was observed with a moderate dose of challenge Ag (32). B6
background mice are hyporeactive to PCl CS and thus were contact sensitized twice on days 0 and 1. Resulting thickness of the
Ag-challenged ears was measured on days 4 or 7 in the case of B6
background mice with a dial caliper (Ozaki MFG Co., Tokyo,
Japan) before challenge and at 2 and 24 h after challenge. Increased ear thickness was expressed as mean ± SE.
DTH against TNP-SRBC (24, 38) was induced in BDF1 mice with TNP-SRBC conjugated by incubating SRBC (5 × 108 cells/ml) with 10 mM
trinitrobenzene sulfate sodium salt in PBS at 37°C for 60 min,
and then washing three times with PBS. For immunization, mice
were injected i.v. with 105 TNP-SRBC on day 0. On day 5, both footpads were challenged s.c. with TNP-SRBC (2 × 108
cells in 40 µl PBS) to elicit DTH, and footpad thickness was measured before and 24 h after challenge.
C3 and C5 activity were titrated by
measuring Ab-dependent C-mediated hemolysis of SRBC by
C3- or C5-deficient human serum (39). In brief, SRBC (5 × 108/ml) were coated with Ab in rabbit antiserum to SRBC. 100 µl of each serum sample or ear extract was mixed with 100 µl of
human C3- or C5-deficient serum diluted 1:20 with gelatin
veronal buffer (pH 7.4), and with 50 µl of Ab-coated SRBC (5 × 108), followed by incubation for 60 min at 37°C. Then hemolyzed supernatants were collected and OD measured at 405 nm.
In Vitro Evaluation of Chemotactic Activity in Ear Extracts.
Ears
that were the site of CS reactions were removed at the base and
three punch biopsies were collected from the distal portion. Biopsies were 12.5 mm2 and were frozen rapidly in liquid N2. Either the whole ear or three punch biopsies per ear were extracted
in 300 or 500 µl cold PBS, with a tissue homogenizer (Biospec
Products, Racine, WI) on ice, followed by centrifugation at
14,000 rpm for 15 min to obtain the supernatant of extracts. Protein concentration was determined by BCA-protein determination kit (Pierce, Rockford, IL). C5 content was evaluated by the
C5-titration assay.
production from NK cells (2), or via IL-12 from macrophages (3),
and IL-4 from NK1.1 CD4+ T cells (4). Furthermore, C
participates in acquired augmentation of B cell Ab responses when C3d is conjugated to Ag (5). This was particularly important when the immunizing Ag was limiting (6,
7). Also, C can participate in elaboration of anaphylatoxins (C3a and C5a), (8), which activate various cell types, as
well as via formation of the activating terminal C5b-9
complex on target cell surfaces (9). Although a negative
regulatory role of C in cellular immunity was suggested recently by demonstrating that cross-linking of membrane
cofactor protein (CD46), led to suppressed IL-12 production (10), the role of C in positive regulation of acquired
cellular immunity such as T cell responses like contact sensitivity (CS)1 and delayed-type hypersensitivity (DTH) (11,
12) has not been understood fully.
(19, 20), TNF-
(20, 21), and migration inhibitory factor (22), to locally recruit and activate
nonspecific bone marrow-derived inflammatory leukocytes
(neutrophils and monocytes) (13).
, which is the
principle Th-1 cytokine of CS/DTH, and also mediates
subsequent cell infiltration. Additional studies in B cell-
deficient mice suggest that B cell-derived immunoglobulin
may be important in early activation of C. Thus, we suggest that C5, possibly derived from Ab fixation of C, played
an important role in the early initiating and subsequent late
T cell-dependent aspects of these cutaneous immune responses.
70°C, thawed, and then incubated with sterile 5 mg/
ml zymosan at 37°C for 60 min to activate C, followed by centrifugation at 14,000 rpm for 10 min. Supernatant was used as zymosan activated mouse serum (ZAMS). sCR1 was a gift from T
Cell Sciences, Inc. (Needham, MA). Mouse anti-murine C5
mAb (BB5.1) and isotype (IgG1)-matched mouse anti-human C8
mAb (135.8) were purified by protein A column from ascites
generated in nude mice injected i.p. with appropriate hybridoma
cells.
In Vitro Quantitative Measurement of IFN- in Ear Extracts.
A quantitative sandwich ELISA for IFN-
used two specific mAbs
(PharMingen, San Diego, CA). In brief, wells were coated overnight with 2 µg/ml capture mAb (R4-6A2) in 0.1 M NaHCO3
(pH 8.3) at 4°C. After blocking with 1% BSA in PBS at room
temperature for 2 h, samples and dilutions of standard recombinant mouse IFN-
(Genzyme Corp., Cambridge, MA) were added
and incubated for 1 h at room temperature. Since there was no
significant difference in IFN-
content between ears extracted
with or without proteinase inhibitor cocktail, which included
PMSF, EDTA, leupeptin, E-64, and pepstatin A, cold PBS alone
was used to extract ears. Then, 1 µg/ml of the other biotinylated
anti-IFN-
mAb (XMG1.2), and 1:3,000 diluted horseradish
peroxidase-conjugated streptavidin (Vector Labs., Burlingame,
CA), were added to probe for IFN-
. Peroxidase substrate (TMB
tetramethylbenzidine) and TMB one component stop solution
(Kirkegaard & Perry Labs, Inc., Gaithersburg, MD) were used for
color development at 450 nm.
Histological Evaluation of Cell Infiltration in CS Ear Responses. 5-µm sections of formalin-fixed, paraffin-embedded ear tissue were stained with hematoxylin and eosin. Semiquantitative evaluation of infiltrating cells per area (1, no infiltration; 2, slight infiltration; 3, modest infiltration; 4, strong infiltration), and also formation of intraepidermal abscesses (1, not seen; 2, few seen; 3, many seen) were determined. Histology in ears was read by an observer blinded as to the experimental design and assigned a numerical grade, from which group means and SE were calculated.
Statistics. Statistics were performed using the two-tailed Student's t test and P <0.05 was taken as the level of significance. Each experimental group consisted of four to six mice.
sCR1 is a newly
developed C-blocking reagent (33) which enabled us
to investigate when C acts in the efferent elicitation phase
of CS. When sCR1 was injected systematically 30 min before Ag challenge in immunized CBA/J mice, both early
(2-h) and late (24-h) components of CS ear swelling responses were reduced significantly (Fig. 1 A, group D vs
C). Since both 2- and 24-h ear swelling responses were affected by sCR1 treatment, the decreased 24-h response
may have been due to effects of sCR1 on the early 2-h events of CS, which are required for elicitation of the 24-h
ear swelling responses (14, 15). Thus, when sCR1 was
given 3 h after Ag challenge, and thus after the required
early events, there was no effect on 24-h ear swelling responses (Fig. 1 A, group E), suggesting that required C
acted early, before 3 h, in the elicitation of CS.
To determine whether another system of DTH responses was similarly dependent on C, acting in the early phase, BDF1 mice were immunized i.v. with TNP-SRBC and then hind footpads were challenged with TNP-SRBC on day 5. Again, sCR1 treatment 30 min before (Fig. 1 B, group D vs C), but not 3 h after Ag challenge (group E) inhibited DTH. Taken together, these results suggested that classical 24-h CS and 24-h DTH, in two different strains of mice, actually depended on C-mediated early events.
C in the Local Site Is Important for CS Elicitation.We reexamined whether CVF, which generally is used to deplete C, would inhibit CS. CVF (10 µg) in PBS was injected i.p. twice per day on days 1 and 2 after immunization. This protocol was reported to exert minimal side effects such as decreased platelets (12, 40). This CVF treatment regimen depleted serum C3 and C5, because C3 was undetectable at 24 and 48 h after CVF treatment, and C5 was decreased to 12.5 and 25% of normal, respectively. Platelet counts of saline versus CVF-injected mice were not different 48 h after the final CVF injection. When sensitized mice were challenged with a conventional, high dose of 0.8% PCl, there were no significant differences between saline- and CVF-injected groups (79% of saline control, P = 0.24). However, when the eliciting dose of PCl was decreased to 0.4% to elicit weaker CS, CVF diminished 24-h ear swelling responses (48% of saline control, P <0.05) (data not shown).
We hypothesized that effects on local C levels might explain these observations with CVF. Thus we attempted to
deplete C locally by directly injecting CVF into ears. CVF
was locally injected into ears 48 h before local Ag challenge, since CVF injected alone s.c. into ears caused no
swelling 48 h later. We found that both 2- and 24-h ear
swelling responses were decreased significantly by local CVF
in CBA mice (Fig. 2 A, group D versus C). In similar DTH
experiments which used BDF1 mice, local preinjection of
CVF into footpads 48 h before Ag challenge also decreased
DTH against TNP-SRBC (Fig. 2 B, group D versus C).
Thus, these experiments showed that C depletion with local CVF could inhibit CS and DTH, and pointed to a role
of local C activation in CS initiation.
Since C depletion by CVF probably was accompanied
with generation of anaphylatoxins such as C3a and C5a,
which could have confused results, we alternately employed anti-C5 mAb to deplete C5. When anti-C5 mAb
was injected i.p. 24 and 4 h before Ag challenge, a negligible amount of C5 remained in serum, as detected by a C5
in vitro titration assay (data not shown). This systemic
treatment with anti-C5 mAb significantly inhibited both
2- and 24-h ear swelling responses (Fig. 3 A, group D versus
C). Importantly, we also injected anti-C5 mAb locally into
ears to deplete C5 at the site of CS. This resulted in decrease of both 2- and 24-h ear swelling responses (Fig. 3 B,
group D versus C). Taken together, these results indicated
that C and particularly C5, acting early at the local CS site,
were important for mediating elicitation of CS responses.
C-dependent Production of Local Chemotactic Activity in CS Ear Extracts.
To determine the mechanism of C participation in CS, we extracted CS ears, which were obtained
24 h after Ag challenge with cold PBS. Total extractable protein was increased in CS, paralleling macroscopic ear swelling measurements 24 h after Ag challenge (Fig. 4 A), and
C5 activity in a C-function assay increased significantly in
CS responses (Fig. 4 B) but not in controls. We then measured local macrophage chemotactic activity in vitro in CS
ear extracts by use of migrating J774A.1 macrophages. We
detected increased macrophage chemotactic activity in the
CS ear extracts, compared to nonimmune controls (Fig. 5,
A and B). To investigate C dependency of this chemotactic
activity found in the local ear site, mice were treated systemically with anti-C5 mAb and significant decrease was
obtained (Fig. 5 A, right). Furthermore, systemic treatment with sCR1 also diminished macrophage chemotactic activity in CS ears (Fig. 5 B, right), confirming that locally extracted macrophage chemotactic activity was C dependent.
These results suggested that either C5a or other C-regulated, locally derived chemotactic factors were responsible
for macrophage chemotactic activity in local CS reactions.
C-dependent Production of Local IFN-
Since IFN- is a crucial cytokine in CS responses
(19), we also quantified IFN-
in CS ear extracts. Increased
IFN-
was found in CS ear extracts 24 h after Ag challenge
(Fig. 6, A and B). Since little connection is usually recognized between C and cytokines, it was of interest to note
that both systemic and local treatment with anti-C5 mAb
significantly inhibited IFN-
production in 24-h CS ears
(Fig. 6, A and B). Thus, C5/C5a was also suggested to regulate local production of IFN-
, which is an important
Th-1 cytokine for elicitation of CS responses.
C-dependent Cell Migration into CS Sites.
Finally, to determine whether cell infiltration as well as macrophage
chemotactic activity (Fig. 5), and IFN- production (Fig. 6),
were decreased by C5 depletion, we examined histology of
CS ear sections. Decreased local cell infiltration in CS ears (Fig. 7, d versus c) and also inhibited intraepidermal abscesses formation (Fig. 7, f versus e) were noted in mice
treated with anti-C5 mAb. Semiquantitative histologic observation was carried out blindly and indicated that C5 depletion resulted in significant decreases in leukocyte infiltration of CS (Fig. 8 A), and in decreased formation of
intraepidermal abscesses (Fig. 8 B).
Impaired 24-h CS in B Cell-deficient Mice.
A central question concerns the mechanism of C activation early in the
cascade of events which leads to T cell infiltration, and then
local production of chemotactic activity as well as IFN- production. We tested CS responses in actively sensitized B
cell-deficient µMT mice which have a deletion in the transmembrane portion of surface IgM, and as a consequence
display an absence of all mature B cells (41). Fig. 9 shows
that 24-h CS was induced in positive control C57Bl/6 mice
(group B) versus unsensitized controls (group A). C57Bl/6
are known to have CS hyporeactivity, compared to, e.g.,
CBA/J mice (for CBA/J see Figs. 1 and 2, group C, at 24 h). Fig. 9 presents similar unsensitized (group C) versus sensitized (group D) groups in C57Bl/6-Igh-6 (µMT) mice,
showing no induction of 24-h CS in immune group D
versus nonimmune group C. There was a significant decrease in 24-h CS in µMT mice (group D) compared to
the positive C57Bl/6 controls (group B). The 2-h macroscopic responses were too small for conclusions about statistical significance.
These results demonstrated a defect in elicitation of CS
in B cell-deficient mice. This may signify that antibodies
are responsible for C activation early in CS, and for subsequent derivation of C-related chemotactic factors which
help recruit T cells, later produce IFN-, and generate the
late 24-h inflammatory infiltrate.
Our results point to a previously unrecognized role of C
in CS and DTH which occurs during early required events
in the efferent phase of these classical T cell-mediated immune responses. The early events of CS are characterized by
processes that are required to recruit Th-1 cells to local sites
of Ag challenge to initiate subsequent classical "delayed"
aspects of these in vivo immune inflammatory reactions by
producing Th-1 cytokines such as IFN-. Although these
late steps in the Th-1 cell-dependent inflammatory cascade
of CS and DTH have been well described (13), the processes that lead to tissue Th-1 cell recruitment are not fully
understood. Recombinant sCR1 is a recently developed
C-blocker which enabled us to distinguish an early versus
late locus of C in elicitation of CS responses by administration 30 min before, compared to 3 h after, local Ag challenge. Thus, only when sCR1 was given 30 min before Ag
challenge were CS and DTH responses inhibited, while giving sCR1 3 h after challenge produced no effect (Fig. 1).
Thus, it appeared that after Ag challenge, C was involved
in 0-3-h early events which were needed to elicit 24-h CS.
In contrast to sCR1, systematic CVF depletion of C failed to demonstrate an involvement of C in CS and DTH, when a high amount of Ag was used for sensitization and challenge (11, 12). However, it was shown previously in studies of Ab production that C participation was more evident when the amount of Ag was limiting (6, 7). Similarly, in congenitally C5-deficient mice (32), we demonstrated previously that CS was impaired when moderate Ag doses were used for sensitization or elicitation. Thus in this study, we reexamined the effect of C depletion by CVF by using a moderate eliciting Ag dose and found impaired CS. We therefore propose that elicitation of CS with a large Ag dose can overcome CVF-induced, or congenital C depletion, whereas C is required to elicit CS when moderate Ag doses are employed. An explanation for this may be that a large Ag dose triggers induction of elicitation of CS via early events that are in part due to nonspecific early irritation and nonspecific inflammation and that probably produce vascular activation and permeability. In fact, it was shown recently that an irritative chemically reactive hapten such as TNP (PCl) contributes nonspecifically to elicitation of Ag-specific CS (42). Thus, specific and nonspecific effects of a given Ag and the dose need to be taken into account in deciding the pathogenesis of the early initiating aspects of CS.
C components are produced by multiple cell types (43- 46). Local tissue macrophages were proposed as the C source in a DTH model associated with Listeria protection (47). Thus, C5-deficient mice which were locally reconstituted with normal C5-producing tissue macrophages were protected against Listeria but were not protected with macrophages from C5-deficient donors (47). Therefore in this study, we treated mice with CVF locally into the ears to examine the role of local C in CS. We found that both 2- and 24-h CS responses were decreased significantly by this local depletion of C (Fig. 2), suggesting that locally produced C was involved in the crucial early events required in CS and DTH.
There are limitations to the use of CVF due to its intrinsic generation of C3a and C5a anaphylatoxins and possible alteration in platelets which are known to be involved in CS (15, 16). Thus, we also used anti-C5 mAb to deplete C5 (36, 37), but without these complications. Systemic anti-C5 mAb treatment strongly inhibited both 2- and 24-h CS ear swelling responses (Fig. 3 A, group D versus group C). Further, local injection of anti-C5 mAb directly into the ears, also profoundly inhibited both 2- and 24-h CS responses (Fig. 3 B, group D versus group C), similar to local depletion of C produced by CVF (Fig. 2). These results strongly indicated that local C, possibly C5, is required early for eliciting CS, and that the trigger for activation of C in CS occurs locally.
To directly analyze events in CS, we developed a technique to extract macromolecular components from the ears of CS ear reactions. We found an increase in chemotactic activity for macrophages at the local CS sites, that was decreased by systemic administration of anti-C5 mAb, or by sCR1 (Fig. 5). C5-derived C5a itself, or possible other C-induced chemotactic factors, may be responsible for the observed chemotactic activity in CS ear extracts. Since migration inhibitory factor (22) and IP-10 (48), both of which are chemotactic, have been reported to be upregulated in sites of DTH reactions, further study is necessary to discriminate C5a from these other macrophage chemotactic factors. However, since we detected increased C5 in 24-h CS ear extracts (Fig. 4 B), potent chemotactic C5a may have been generated by newly arrived C5, in addition to locally generated C5a which was suggested by treatment with local CVF, and also local anti-C5 mAb.
Important additional findings were made regarding IFN-
in CS ear extracts. Since IFN-
is a crucial Th-1 cytokine
in DTH (19), we also measured IFN-
in local CS ear sites
and as expected found increased local levels at 24 h. Since
little connection generally is recognized between C and cytokines, we were surprised to find that the increased 24-h
IFN-
production in CS ears was decreased significantly by
C5 depletion, either systemically or locally, following treatment with anti-C5 mAb, which has a locus early in elicitation of CS (Fig. 6). In addition, we also performed time course
experiments to quantitate IFN-
by sandwich ELISA and
semiquantitative reverse transcription-PCR for IFN-
mRNA in CS ears. These studies showed that neither IFN-
nor its mRNA was detected at CS sites for up to 4 h after
Ag challenge, when the early CS-initiating events were
completed (Tsuji, R.F., unpublished observations). At this
time, Th-1 cells probably were not yet fully recruited and
locally activated, but C activation was complete. In further
studies, we noted that immunodeficient TCR-
knockout mice showed no IFN-
production in CS ear extracts at
any time. Taken together, these observations strongly suggested that IFN-
production in local CS ear sites was due
to late arriving Ag/MHC-specific
/
TCR+ CS-effector
Th-1 cells. Therefore, anti-C treatment via anti-C5 probably impaired the early phase, C-dependent recruitment of Th-1 cells into the local CS site, resulting in decreased later production of IFN-
at these local CS sites.
Since Th-1 cell recruitment and activation for subsequent IFN- production should lead to local recruitment
of inflammatory leukocytes, we performed histology to determine the possible C dependency of the characteristic cell
infiltration in CS. Accordingly, we found that anti-C5
mAb treatment reduced the numbers of infiltrating leukocytes (Figs. 7, d versus c, and 8 A). In particular, anti-C5 mAb treatment led to drastic decreases in characteristic intraepidermal abscesses in 24-h CS ears (Figs. 7, e versus f,
and 8 B). This apparent C5 dependency of leukocyte infiltration was thus a histology analogue which was consistent
with our finding of decreased macrophage chemotactic activity in 24-h CS ear extracts (Fig. 5). Although we did not
directly study the effect of C on Th-1 cell recruitment, decreased local production of IFN-
in CS ears of anti-C5
mAb-treated mice (Fig. 6) suggested that recruitment of
Th-1 cells was decreased. Thus, the inhibited leukocyte infiltration may have been due to decreased C-dependent
Th-1 recruitment into CS ears. We noted previously that
treatment with specific antiplatelet Ab led to depletion of
platelets, a potential source of local 5-HT able to activate
endothelium for Th-1 recruitment, and also led to marked
decrease in formation of intraepidermal abscesses in CS (15). Thus, the action of C in CS might involve intermediate platelet and mast cell activation, perhaps via C5a acting
on their C5a receptors, with subsequent 5-HT release,
contributing to Th-1 cell recruitment and then leukocyte
arrival.
The exact function of C5 acting early in elicitation of CS
responses is not known. There are two potential C5-derived
mediators such as anaphylatoxin C5a, and the terminal
C5b-9 complex, generated after C5 cleavage, and also a
potent cell activator in sublytic amounts (9). One likely
possibility is that either or both of these C-mediators trigger local mast cells and/or platelets via C5a receptors early
in CS, to release 5-HT which is a key mediator of vascular
permeability, and perhaps vasoactivity in the initiation of
CS (29, 31, 49). Alternately, or in addition, these C5-
derived mediators may directly activate the local vasculature to enhance permeability and expression of adhesion
molecules (50, 51), contributing to local T cell and/or leukocyte recruitment. In fact, it was reported that C augments immune inflammation in the lung by upregulating
expression of vascular ICAM-1 (52). Also C5a was shown
recently to be responsible for upregulation of lung vascular
P-selectin (53). In other CS studies, we noted prolonged
C5a activity in vivo when we gave an inhibitor of the C5a-inactivating enzyme, which converts active C5a to inactive C5a desArg (54). There were also augmented CS ear swelling responses, suggesting that endogenous released C5a is
involved in CS elicitation. In addition, experiments with
C5a receptor (/
) mice (55) suggest a role of C5a in CS
(Tsuji, R.F., C. Gerard, and P.W. Askenase, manuscript in
preparation).
It was of interest to find what causes the crucial early C activation in CS responses. We investigated this by using B cell-deficient µMT mice and found that their 24-h CS was decreased (Fig. 9). Taken together, an involvement of B cells and therefore probably of immunoglobulins in CS responses is suggested, but awaits further detailed demonstration. These findings suggest a heretofore unappreciated crucial role of B cell-derived specific Ab in the early initiating phase of T cell-mediated immunity in vivo, as exemplified by CS and DTH. Current studies suggest that IgM is an important involved immunoglobulin isotype and may be produced by the B-1 subset of B cells. (Tsuji, R.F., and P.W. Askenase, manuscript in preparation).
In summary, this study shows that local C activation is
involved in crucial early initiating events which are required to elicit CS and DTH. This may be due to C activation triggered via B cell-derived immunoglobulin, and
subsequent local generation of C5a and/or the terminal
C5b-9 complex, leading to local production of C-related
chemotactic factors such as C5a, which facilitate local recruitment of Th-1 cells to then interact with APC to produce IFN- at the local site of CS/DTH to recruit an inflammatory infiltrate. These data provide a new example of
an important augmenting role of C, which is usually regarded as a mediator of innate immunity but here acts as an
initiator in an early required phase of acquired T cell immunity in vivo.
Address correspondence to Dr. Ryohei F. Tsuji, Noda Institute for Scientific Research, 399 Noda, Noda-shi, Chiba-ken 278, Japan. Phone: 81-471-23-5573; FAX: 81-471-23-5550.
Received for publication 21 January 1997 and in revised form 20 June 1997.
The authors are indebted to Dr. Lindsay N. Donald of T Cell Sciences, Inc. for the kind gift of sCR1. We thank Drs. Peter J. Lachmann, Tony Hugli, Vipin Paliwal, and Rajani Ramabhadran for their valuable advice. We are also grateful to Marilyn Avallone for her superb secretarial skills.This work was supported in part by grants from the National Institutes of Health to P.W. Askenase (AI-12211, AI-26689, and AI-07174).
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