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Departments of
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Pediatrics,
Surgery,
Pathology, and
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Immunology, Duke University Medical Center, Durham, NC 27710;
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Department of Surgery, The Mayo Foundation, Rochester, MN 55905;
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Department of Pathology, Veterans Affairs Medical Center, Durham, NC 27710; and
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Avant Immunotherapeutics, Inc., Needham, MA 02494
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To this point, several pathways for lysis of cells involving C1 and components of the alternative pathway have been described. Some years ago, we described a pathway that we termed at that time the C1-bypass pathway. This activation pathway utilized Ab, C1, and components of the alternative (or bypass) pathway to lyse erythrocytes and was functional in the complete absence of C4 (2, 5, 6). This bypass pathway was active in C4-deficient guinea pig serum. Given that the alternative complement pathway is no longer termed the bypass pathway, this term is misleading and the pathway is better referred to as the C4-bypass pathway, because it bypasses C4. We will call it such. Some years later, a C2-bypass pathway that utilizes Ab, C1, C4, and components of the alternative pathway was observed to be operative in C2-deficient human serum, thereby bypassing C2 (3, 4, 7). It was felt to be quite similar to the pathway we had described earlier. Interestingly, a C4-bypass pathway was also reported in a different experimental model, namely the lysis of IgM-sensitized Giardia lamblia trophozoites by C2-deficient human and C4-deficient guinea pig sera (8).
Despite isolated reports suggesting the possible involvement of a C4-bypass complement activation pathway (9, 10) and a C2-bypass pathway (11) in humans, the in vivo relevance of these in vitro phenomena has never been clearly addressed. In the present study, tissue damage mediated by Ab and complement was examined in guinea pigs that are genetically deficient in the second and fourth components of complement. Normal guinea pigs were used as control animals. The immunopathologic model studied was Forssman shock, a well-studied reaction in guinea pigs. The injection of rabbit IgG anti-Forssman Ab into guinea pigs leads to a cataclysmic reaction, with rapid pulmonary edema and hemorrhage leading to death. We have previously shown that the reaction is absolutely dependent on activation of the classical complement pathway and cannot be induced in C4-deficient guinea pigs (12). However, to our surprise, unlike C4-deficient guinea pigs, when C2-deficient guinea pigs were challenged with rabbit anti-Forssman IgG Ab, pulmonary shock occurred with pathologic findings that resembled those in normal guinea pigs. We have analyzed the difference in this immunopathologic reaction between C2-deficient and C4-deficient animals in detail in both in vivo and in vitro studies. This appears to represent the first example of in vivo involvement of the C2-bypass pathway of complement activation in the induction of tissue damage.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Isotonic Veronal-buffered saline (VBS)3 containing either 0.5 mM MgCl2, 0.15 mM CaCl2, and 0.1% gelatin (GVBS2+) or 10 mM EDTA and 0.1% gelatin (EDTA-GVBS) as well as low ionic strength dextrose-GVBS2+ (DGVBS2+) were prepared as described (13). Isotonic GVBS2+ containing 8 mM EGTA and 5 mM MgCl2 (EGTA-GVBS) for alternative pathway activation assays was prepared as described (14). In some experiments, EGTA-GVBS contained 20 mM EGTA to ensure maximal calcium ion chelation in highly concentrated serum.
Sheep blood was collected aseptically in Alsever’s solution. After removal of the buffy coat and several washes in EDTA-GVBS, GVBS2+, and 60% DGVBS2+, the erythrocytes were standardized to 1 x 109/ml and stored at 4°C in DGVBS2+. For alternative pathway assays, blood was collected from normal New Zealand white rabbits into disodium EDTA and processed as sheep blood.
Blood samples were collected from normal Hartley, C2-deficient, and C4-deficient guinea pigs via cardiac or cranial vena cava puncture under anesthesia with isoflurane. Serum was prepared from these blood samples using standard methods and immediately frozen at -80°C. Sera were adsorbed twice with packed fresh sheep erythrocytes or fresh rabbit erythrocytes on ice for 30 min to remove natural Abs.
Hemolytic assays
The ability of sera from C2-deficient, C4-deficient, and normal guinea pigs to lyse Ab-coated sheep erythrocytes was assayed as follows. Sheep erythrocytes at a concentration of 5 x 108cells/ml were sensitized with various dilutions of either a rabbit anti-boiled sheep erythrocyte stroma IgM-containing antiserum or a rabbit anti-boiled sheep erythrocyte stroma IgG fraction. It is known that virtually all of the hemolytic Ab in these preparations reacts with purified Forssman glycosphingolipid. The antiserum used in our experiments contained anti-sheep red blood cell stroma IgM Abs with undetectable IgG hemolytic reactivity (data not shown). Sensitized cells (25 µl at 1 x 109/ml) were incubated at 37°C for 1 h in the presence of various dilutions of guinea pig serum in a total volume of 75 µl. Where indicated, inhibitors of complement were added to the reaction mixture. The reaction was stopped by adding 2 ml of ice-cold EDTA-GVBS, the tubes were spun, and the extent of hemolysis in the supernatant was read at 412 nm. Total hemolytic complement activity (13) and alternative pathway activity (14) assays were performed as described.
Complement inhibitors
To assess the role of both the classical and alternative pathways of complement activation in our in vitro and in vivo assays, soluble forms of complement receptor type 1 (CR1) were used. Soluble CR1 (sCR1), lacking the cytoplasmic and transmembrane domain of CR1, and a soluble recombinant form of sCR1, lacking the first of the four long homologous repeat sequences (LHR-A), which contains one of the three moderate affinity binding sites for C4b (sCR1[desLHR-A]), were provided by Avant Immunotherapeutics (Needham, MA). sCR1 blocks C3 and C5 convertases of both the classical and the alternative complement pathways (15). Recently, sCR1[desLHR-A] was produced and shown to block preferentially the alternative pathway (16). LHR-A contains one of the three moderate-affinity C4b binding sites. Absence of LHR-A, within the engineered molecule, abrogates control of the classical pathway C3 convertase. Thus, the classical pathway remains intact, whereas alternative pathway function is blocked. In in vivo experiments, both of these agents were injected i.v. at a dose of 15 mg/kg body weight (17).
Animals
Normal Hartley guinea pigs (400–800 g) were purchased from Charles River Laboratories (Wilmington, MA), and guinea pigs deficient in the second (C2) and fourth (C4) components of complement (500–800 g) were from colonies housed at the Duke University Vivarium (Durham, NC). Both C2-deficient (18) and C4-deficient (19) guinea pigs were derived from the original colonies. All animals used in our experiments were devoid of either functional C2 or functional C4 activity according to well-established and highly sensitive hemolytic assays (20, 21).
Forssman shock experiments
Details relative to the preparation and characterization of rabbit anti-boiled sheep erythrocyte stroma Abs are as described (12). An IgG fraction from rabbit antiserum collected several weeks after primary immunization was prepared according to standard methods (22). Heat inactivation (56°C, 30 min) of all sera before Ab purification was performed to ensure absence of hemolytically active complement. Hemolytic titration of Ab preparations in the presence of excess complement was conducted as described (23). Both the IgM-containing antiserum and the IgG fraction were used in in vitro assays, whereas only the IgG fraction was used in in vivo experiments, because only anti-Forssman IgG Abs are capable of inducing the Forssman shock reaction (24). A total of 0.5 ml IgG anti-Forssman Ab injected i.v. proved to be sufficient to cause the classical symptoms associated with Forssman shock (12) and uniformly led to pulmonary shock and death within 10 min after injection in normal Hartley guinea pigs of 350–500 g body weight. A volume of 1 ml induced a similar reaction in 800-g guinea pigs. Briefly, animals were injected via either the lateral saphenous vein or one of the branches of the cephalic vein and observed until reactions occurred. In experiments in which complement inhibitors were used, a catheter was inserted into a branch of one of the cephalic veins while the animal was under isoflurane anesthesia. sCR1 or sCR1[desLHR-A] was injected to achieve a final dose of 15 mg/kg body weight. Endotoxin-free sterile PBS (Life Technologies, Grand Island, NY) served as a control (vehicle). Fifteen minutes later, anti-Forssman IgG was injected. Blood samples were collected before inhibitor or Ab administration and when severe respiratory distress, which preceded death, was observed. In cases in which no reaction was observed, animals were bled and killed 60–90 min after Ab injection.
Tissue sampling and processing
At the animal’s death, the lungs were rapidly removed. Small sections were embedded in OCT medium, quick-frozen in liquid nitrogen-precooled isopentane, and stored at -80°C until further analysis. Sections (5 µm) of the frozen tissues were cut and tested by immunofluorescence for the presence of rabbit IgG, guinea pig C3, and guinea pig C4. FITC-labeled goat anti-rabbit IgG (IgG fraction) and FITC-labeled goat anti-guinea pig C3 (IgG fraction) were purchased from ICN Biomedicals (Costa Mesa, CA). A FITC-labeled nonimmune goat IgG fraction (ICN Biomedicals) was used as a negative control. A polyclonal Ab directed against guinea pig C4 was prepared in our laboratory by immunizing C4-deficient guinea pigs with normal guinea pig serum. An IgG fraction was prepared that reacted with guinea pig C4 on the basis of immunodiffusion. Because of its weak reactivity toward guinea pig C4, this Ab preparation was used in immunofluorescence studies using a two-step amplification system. The IgG fraction was labeled with biotin (Sigma, St. Louis, MO), and its binding to guinea pig lung tissues was evidenced using streptavidin-FITC (Jackson ImmunoResearch, West Grove, PA). An IgG fraction from a nonimmune C4-deficient guinea pig serum was prepared and labeled with biotin and used as a negative control. Abs to guinea pig C4 and C3 were adsorbed with normal rabbit serum, whereas the anti-rabbit IgG was adsorbed with normal guinea pig serum to avoid nonspecific reactivity.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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As expected from earlier studies (4, 5), lysis of
Ab-sensitized sheep erythrocytes was noted when high concentrations of
both C2-deficient and C4-deficient guinea pig sera were used (Fig. 1
). Ab isotype and the density of Ab
molecules on the surface of the target cell also played a crucial role
in the complement-mediated lysis of these cells. Dose-response curves
were established for lysis of IgG and IgM Ab-sensitized sheep
erythrocytes in the presence of excess C4-deficient and C2-deficient
guinea pig serum. Whereas sera from C2-deficient guinea pigs lysed both
IgM- and IgG-sensitized sheep erythrocytes (Fig. 1, A and
B), in general, more efficient lysis was observed when IgG
was used as a sensitizing agent (Fig. 1B). C4-deficient
guinea pig serum proved much less efficient than C2-deficient guinea
pig serum at lysis of sheep erythrocytes at all concentrations of Ab.
Sera from C4-deficient guinea pigs induced lysis of IgG-sensitized
cells, whereas the effect on IgM-sensitized cells was negligible (Fig. 1, C and D). Maximal lysis of cells was seen with
high concentrations of sensitizing Ab and high concentrations of C2-
and C4-deficient sera. The observed lysis of erythrocytes was
complement dependent, as incubation in the presence of EDTA in all
cases decreased specific lysis to <5% in cases in which lysis in the
absence of EDTA was maximal (data not shown). Heat inactivation of
serum samples at 56°C for 30 min abolished their lytic activity.
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To assess the relative role of the classical and alternative
pathways of complement activation in the lysis of heavily sensitized
sheep erythrocytes by C2-deficient and C4-deficient guinea pig sera, a
buffer containing 20 mM EGTA and 5 mM MgCl2 was
used to allow only the activation of the alternative complement
pathway. Cells were sensitized with a 1:125 dilution of either IgM or
IgG anti-Forssman Ab. These were the highest concentrations of Ab
that could be used without agglutination of cells. As shown in Fig. 2, EGTA treatment, which blocks C1
function, markedly impaired the lysis of both IgM- (Fig. 2A)
and IgG-sensitized (Fig. 2B) erythrocytes by C2-deficient,
C4-deficient, and normal guinea pig sera, indicating that the classical
pathway was utilized in these sera. Treatment of the various sera at
50°C for 30 min, which destroys factor B activity, impaired the
ability of C2-deficient and C4-deficient guinea pig sera to lyse
sensitized sheep erythrocytes, whereas normal guinea pig serum was
unaffected by such treatment (Fig. 2, A and B),
indicating that the alternative pathway contributed to lysis in
C2-deficient and C4-deficient sera.
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A). In sharp
contrast, both sCR1 and sCR1[desLHR-A] inhibited the lysis of heavily
sensitized sheep erythrocytes by C2-deficient and C4-deficient guinea
pig sera in a dose-dependent fashion. On a molar basis, a higher
concentration of sCR1[desLHR-A] than sCR1 was required to achieve the
same extent of inhibition of lysis (Figs. 3, B and
C). In all cases, substitution of complement inhibitors by a
control protein (human serum albumin) induced no significant change in
the percentage of lysis induced by serum alone (data not shown). These
results confirm that the alternative pathway is necessary for lysis of
sensitized sheep erythrocytes by C2-deficient and C4-deficient guinea
pig sera. In addition, our data show for the first time that sCR1 and
sCR1[desLHR-A] derived from human CR1 have the ability to interfere
with guinea pig complement activation, as they do with human
complement. However, there are no published data to indicate that human
CR1 has all of the functional activities in guinea pig serum that exist
in human serum.
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Normal, C2-deficient, and C4-deficient guinea pigs were subjected
to Forssman shock according to well-established methods. Observations
are depicted in Table I. All normal
guinea pigs died of pulmonary shock within 2–10 min after injection of
anti-Forssman IgG Ab, as expected. Three C4-deficient guinea pigs
were subjected to the same treatment, but no adverse effects were seen
at up to 70 min of observation after Ab administration. Macroscopic
observation of the lungs after the animals were sacrificed showed no
signs of pulmonary damage or hyperinflation, which is the hallmark of
Forssman shock. In striking contrast, C2-deficient guinea pigs
demonstrated sensitivity to the i.v. injection of the anti-Forssman
Ab with reactions appearing in a delayed fashion as compared with
normal guinea pigs. Three animals died of pulmonary shock within 10–30
min after challenge with the Ab. Three other guinea pigs experienced a
sublethal reaction observed as respiratory distress without death. In
the latter case, the lungs were markedly hyperinflated, as were those
of normal guinea pigs with Forssman shock. In cases in which the shock
was lethal, blood-tinged sputum obstructing the airways was observed,
as is observed in complement-sufficient guinea pigs. Control
experiments in two normal guinea pigs injected with a similar amount of
preimmune rabbit IgG fraction failed to demonstrate any pulmonary
reaction. The dependence on complement of the reaction observed in
C2-deficient guinea pigs was tested by treatment of two animals with
cobra venom factor (CVF). As observed in normal guinea pigs, treatment
with CVF completely eliminated any reaction to the i.v. injection of
rabbit anti-Forssman IgG (data not shown). Two C2-deficient animals
were also injected i.p. with normal guinea pig citrate plasma as a
source of C2 and were subjected to Ab challenge 18 h later. Even
though the functional C2 levels in the blood of each animal roughly
approximated 5% of the activity found in the injected material, a
normal pulmonary reaction followed by death within 5 min after Ab
injection was observed in C2-reconstituted C2-deficient guinea pigs
(not shown).
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Because the pulmonary reaction we observed in C2-deficient guinea
pigs was most unexpected, given that the Forssman reaction is strictly
dependent on the classical pathway of complement activation (as proven
by the lack of a reaction in C4-deficient guinea pigs) and C2-deficient
animals have total absence of C2, we conducted experiments to assess
the role of both pathways of complement activation in this reaction. We
used sCR1 to block both the classical and the alternative complement
pathways and sCR1[desLHR-A] to block the alternative complement
pathway. In standard hemolytic assays, administration of sCR1
completely inhibited complement activity via the classical and
alternative pathways. Administration of sCR1[desLHR-A] resulted in
complete inhibition of the alternative pathway but not of the classical
pathway. Representative examples are given in Fig. 4. It is noteworthy that blood samples
were taken after Forssman shock. Some depression of serum complement
titers is noted compared with preshock samples in animals in which
there was no complement inhibition. Reactions observed in each animal
are described in Table II. As expected,
sCR1 protected both normal guinea pigs and C2-deficient guinea pigs
from Forssman shock (Table II). In contrast, normal guinea pigs
injected with sCR1[desLHR-A] before injection of the Ab underwent
pulmonary shock, as did control animals injected with PBS before
anti-Forssman Ab challenge. However, C2-deficient guinea pigs were
protected from Forssman shock when treated with sCR1[desLHR-A] before
Ab challenge. The lungs in these animals appeared normal with no sign
of pulmonary damage.
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Lung sections from normal, C2-deficient, and C4-deficient guinea
pigs were frozen upon the animals’ deaths. Immunofluorescent staining
for the presence of rabbit IgG, guinea pig C4, and guinea pig C3 was
performed. In all animals tested, rabbit IgG deposition was observed in
alveolar septa, primarily in association with alveolar capillaries of
the lung (Fig. 5). C4 deposition was
observed within the lungs of both normal (Fig. 6A) and C2-deficient (Fig. 6B) guinea pigs subjected to Forssman shock. Anti-guinea pig
C4 staining occurred in alveolar septa, mainly in association with
alveolar capillaries. Anti-guinea pig C4 staining, although relatively
weak in intensity, was clearly above that observed with a matched
control Ab (Fig. 6, C and D). However, we were
not able to find differences in the intensity of anti-guinea pig C4
staining in lung tissues from normal and C2-deficient guinea pigs
treated with PBS, sCR1, or sCR1[desLHR-A] before Forssman shock
induction (data not shown). Massive C3 deposition was observed in the
alveolar septa, mostly associated with alveolar capillaries of lung
tissues from normal and C2-deficient guinea pigs either untreated or
treated with PBS before anti-Forssman Ab injection (Fig. 7, A and B). When
both normal and C2-deficient guinea pigs were treated with sCR1 15 min
before Forssman shock induction, the intensity of anti-guinea pig
C3 staining in the lung tissues decreased dramatically (Fig. 7, C and D) to levels comparable with the
fluorescence intensity observed with a matched control Ab (not shown).
When sCR1[desLHR-A] was used as an anti-complement agent, the
intensity of anti-guinea pig C3 staining decreased dramatically
only in C2-deficient guinea pigs (Fig. 7F), whereas it was
intense in normal animals treated with the same agent at the same dose
(Fig. 7E).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Thirteen years after our original observation, Matsushita and Okada (3) observed similar lysis when heavily sensitized sheep erythrocytes were incubated with high concentrations of C2-deficient human serum. Again, C1 was required for lysis, as were components of the alternative pathway. This pathway was termed the C2-bypass pathway. Both IgM and IgG Abs have the ability to activate these unusual complement activation pathways, although different IgM preparations have differing activities on the observed pattern of lysis (6). In isolated reports, the C4-bypass pathway of complement activation was proposed as being involved in human pathologic situations, such as chronic urticaria and angioedema (9), and hemolytic uremic syndrome (10). Using Giardia lamblia trophozoites as a target, Deguchi et al. (8) demonstrated a role of the C4-bypass activation pathway in the lysis of this protozoan parasite. IgM Abs isolated from patients with high lytic activity toward the parasite were shown to activate the bypass pathway in C2-deficient human serum and C4-deficient guinea pig serum. The lytic activity was lost on depletion of C1 or factor B from the serum samples, indicating an involvement of both the classical and alternative pathways of complement activation with no requirement for C4 and C2.
Lysis of sensitized sheep erythrocytes via the C2-bypass pathway depends on alternative pathway function (3). In addition, activation of the alternative pathway leading to erythrocyte lysis in the C2-bypass activation pathway was shown to require the presence of C4b on the sensitized cell rather than C1q (3). By depleting specific complement components, Steuer et al. (4) demonstrated that the lysis of heavily sensitized sheep erythrocytes by C2-deficient human serum depends on activation of C1, C4, and components of the alternative pathway. In binding studies, this group further demonstrated that C4b could bind the alternative pathway C3 convertase via a weak interaction with C3b (7) instead of directly forming a C3 convertase by binding factor B, as was previously proposed (3). Both of the above-cited reports were studies performed with human serum as a source of complement. Our in vitro studies, using C2- and C4-deficient guinea pig sera, are in agreement with the above observations and show for the first time the activity of a C2-bypass pathway in C2-deficient guinea pig serum.
Abs of IgG isotype are more effective than IgM in inducing the lysis of
sheep erythrocytes by complement-deficient sera (Fig. 1
), as was
reported by Matsushita and Okada (3) for C2-deficient
human serum and by us (5) for C4-deficient guinea pig
serum. It is interesting that IgG Ab causes more lytic damage in the in
vitro bypass pathways and is the only Ab isotype capable of causing
Forssman shock (24). The reason why IgG Abs induce
more extensive tissue damage is not clear. However, it should be noted
that rabbit anti-Forssman IgG was shown to induce far more
pore-forming lytic lesions than IgM when guinea pig complement is used
as a complement source (25). The presence of IgG rather
than IgM could favor alternative pathway activation by its ability to
amplify C3b deposition on a target surface (26).
In the studies reported here, C2-deficient guinea pig serum proved to
be more potent than C4-deficient guinea pig serum in the lysis of
heavily sensitized sheep erythrocytes. As shown in Fig. 1, higher
specific erythrocyte lysis was achieved with C2-deficient serum whether
an IgM antiserum or an IgG fraction was used. The lytic process was
dilution sensitive, as high concentrations of serum (1:3 or 1:9) were
required for efficient lysis of the red cells. Conditions for sheep
erythrocyte lysis were compared between C2-deficient and C4-deficient
guinea pig sera in standard complement assays. These assays are
suitable for quantitative analysis of the complement system in various
settings but do not reflect physiologic conditions in which undiluted
plasma, high titers and mixtures of IgM and IgG, and opsonization and
phagocytosis more often than cell lysis are involved in the biologic
effects of complement activation. The conditions required for cell
lysis to occur in C2-deficient guinea pig serum approach those that
might be found in the animal. This may explain why the C2-bypass
pathway is missed in standard assays that utilize dilute guinea pig
serum and optimal Ab concentrations.
Lytic activity in C2-deficient and C4-deficient guinea pig sera in our
in vitro studies depended on both the classical and the alternative
pathways. As demonstrated in Fig. 2, incubation of cells and sera (both
from C2-deficient and C4-deficient guinea pigs) in a calcium
ion-chelating buffer containing EGTA and magnesium ions, which
eliminates classical pathway activation, substantially decreased the
ability of the serum to induce lysis of sensitized cells. Furthermore,
heat inactivation of serum for 30 min at 50°C, which is known to
destroy factor B activity, also impaired markedly the lytic activity of
the serum samples from C2- and C4-deficient animals, suggesting a need
for components of the alternative pathway as well in the lysis of
sensitized erythrocytes.
An additional demonstration of the role of the alternative pathway on
the lysis of heavily sensitized sheep erythrocytes by C2-deficient and
C4-deficient guinea pig sera was afforded by studies using genetically
engineered soluble complement inhibitors. sCR1 with its C4b and C3b
binding domains was capable of blocking classical and alternative
pathway function, as reported (16). sCR1 was capable of
inhibiting the lysis of heavily sensitized sheep erythrocytes induced
by normal, C2-deficient, and C4-deficient guinea pig sera in a
dose-dependent fashion (Fig. 3). sCR1[desLHR-A] was also effective at
inhibiting cell lysis induced by both C2-deficient and C4-deficient
guinea pig sera, although it did so somewhat less efficiently than sCR1
(Fig. 3). sCR1[desLHR-A] lacks one of the three moderate
affinity C4b binding sites (27, 28) and was shown to
inhibit the classical pathway much less efficiently than the
alternative pathway (16). It is therefore a preferential
inhibitor of the alternative pathway. In our hands, sCR1[desLHR-A]
did not block the lysis of Ab-sensitized sheep erythrocytes by the
classical pathway. The fact that it inhibits lysis in C2-deficient
and C4-deficient guinea pig serum argues strongly that components of
the alternative pathway are required for lysis.
We tested the ability of the C2-bypass and the C4-bypass pathways to
mediate immunologic damage in the Forssman shock model. Using
C2-deficient and C4-deficient guinea pigs, we were able to show for the
first time the involvement of the C2-bypass pathway in vivo. As
reported in our earlier studies (12), C4-deficient
guinea pigs had no adverse reaction to the i.v. injection of rabbit IgG
anti-Forssman Abs (Table I). To our surprise, C2-deficient
animals underwent either sublethal or lethal reactions that appeared in
a delayed fashion as compared with normal guinea pigs treated the same
way (Table I). This phenomenon was complement dependent, as animals
treated with CVF to deplete C3 and late-acting components behaved as
C4-deficient guinea pigs. Reintroduction of guinea pig C2 by i.p.
injection of normal guinea pig plasma accelerated the symptoms observed
after i.v. injection of the anti-Forssman Ab, and the animals died
in a time course similar to that observed in normal guinea pigs.
To investigate the role of the classical and alternative pathways in
the development of the Forssman reaction in C2-deficient animals, we
used sCR1 and sCR1[desLHR-A] injected i.v. 15 min before Ab
administration. In normal animals, only sCR1 was potent at blocking the
Forssman reaction. In sharp contrast, both sCR1 and sCR1[desLHR-A]
protected C2-deficient guinea pigs from Forssman shock (Table II),
suggesting that the alternative pathway of complement activation was
recruited in C2-deficient guinea pigs but was not required in normal
animals. The requirement for the classical pathway function can be
deduced by the absence of shock in C4-deficient guinea pigs. Therefore,
components of the classical and alternative pathways appear to be
required in Forssman shock in C2-deficient animals. Our previous
studies have shown that there is no blocking factor in C4-deficient
guinea pigs that might interfere with potential complement-related
activity and purified C4 can restore function to the deficient pathway
in these animals (29).
Our immunopathologic studies using lungs from animals subjected to
Forssman shock revealed similar C4 deposition patterns in normal and
C2-deficient guinea pigs (Fig. 6). The same pattern of C4 deposition
was observed in all animals, irrespective of the treatment regimen
administered before anti-Forssman Ab injection (data not shown). In
contrast, no reaction was observed when this Ab was used on lung
sections from normal and C2-deficient guinea pigs that were not
injected with anti-Forssman IgG or when a matched control Ab was
used. This observation therefore suggests that activation of early
components of the classical pathway up to the missing component
(C2) occurs on IgG-coated cellular targets in vivo in C2-deficient
guinea pigs, ruling out the sole activation of components of the
alternative pathway in the observed phenomenon.
There was massive C3 deposition within the lungs of normal and
C2-deficient guinea pigs that experienced Forssman shock (Fig. 7, A and B). Treatment of both normal and
C2-deficient guinea pigs with sCR1 before induction of Forssman shock
decreased the binding of C3 into the lungs (Fig. 7, C and
D) to levels comparable with those observed with a matched
control Ab. CR1 allows for a faster decay of both the classical and
alternative pathway C3 and C5 convertases and is a cofactor for factor
I-mediated cleavage of C3b to iC3b in humans (30).
These functions of CR1 might explain our data and further support
the strict dependency of Forssman shock on complement activation. When
normal guinea pigs and C2-deficient guinea pigs were treated with
sCR1[desLHR-A], an inhibitor of the alternative pathway and not
of the classical pathway (16), only C2-deficient guinea
pigs survived the shock and showed almost complete absence of C3
deposition within their lungs (Fig. 7F). In contrast, normal
animals treated with the same agent failed to show any decrease in C3
binding in the lung (Fig. 7E) and died of Forssman shock as
untreated animals did. This result demonstrates that C3 deposition and
Forssman shock in C2-deficient guinea pigs are dependent on the
triggering of the alternative pathway. Although this pathway is clearly
not required for Forssman shock to occur in normal guinea pigs, the
possible contribution of this pathway to the reaction in such animals
cannot be ruled out.
As we observed C4 deposition within the lungs of both normal and C2-deficient guinea pigs, we can postulate that in animals lacking functional C2, a C2-bypass pathway can be triggered in vivo to induce tissue damage. As was demonstrated in in vitro experiments, this pathway requires Ab, early components of the classical pathway (C1, C4), and components of the alternative pathway. No obvious quantitative difference in IgG, C4, and C3 binding within the lungs of normal and C2-deficient guinea pigs was noted. Thus, our in vitro findings correlate with our in vivo observations. The C2-bypass pathway can lead to complement-mediated tissue damage but is relatively inefficient. High concentrations of Ab and complement are required to induce lysis of sheep erythrocytes in vitro. In vivo, the clinical signs of Forssman shock occur in C2-deficient guinea pigs in a delayed fashion as compared with normal guinea pigs. Therefore, more complement activation via the alternative pathway is required, which may explain the delay in the occurrence of the symptoms of Forssman shock.
One might postulate that the newly discovered mannan-binding lectin (MBL) pathway plays a role in the C2-bypass pathway, as observed in C2-deficient guinea pigs. MBL can substitute for C1q in activating the classical pathway on binding to microbial carbohydrates. MBL associates with two serine proteases, termed MASP-1 and MASP-2 (31). This pathway was shown to be activated in the presence of sensitizing Ab. However, MBL appears to require IgG that lacks terminal galactose residues to trigger complement activation (32). Normal mammalian IgG has terminal galactose on carbohydrate residues, and the Abs used in this study lacked terminal galactose residues. MBL is reported to activate the alternative pathway on sheep erythrocytes coated with mannan (33). Activation of the alternative pathway in this system is reported to require C4 and C2 of the classical pathway. If these early reports prove correct, it is unlikely that MBL plays a role in the C2-bypass pathway. Nevertheless, as more is learned about the MBL pathway of complement activation, it may be found to play a role in this phenomenon.
The presence of bypass pathways of complement activation may well give insights on the evolution of the complement system, as proposed by Farries et al. (34). It may be that C1 first evolved as a way of allowing Ab to activate the alternative pathway via the C4-bypass pathway. The appearance of C4 would have improved the ability to induce complement activation by Abs bound to a target surface, although with much less efficiency than the classical pathway as currently known. The further addition of C2 to the cascade may have allowed efficient complement activation through the classical pathway.
In summary, we confirm the presence of C1-dependent C4- and C2-bypass pathways in the guinea pig, demonstrate that the C2-bypass pathway is far more damaging immunopathologically, and demonstrate for the first time the ability of the C2-bypass pathway to cause an immunopathologic effect in vivo. The human pathologic relevance of our findings is still not clear, but it is noteworthy that Traustadòttir et al. (11) recently observed that a similar activation pathway depending on C1, C4, and components of the alternative pathway is involved in a model of immune complex binding to red blood cells mediated via CR1. Our results may explain why few C2-deficient patients have recurrent infectious problems. It is known that the classical complement pathway contributes to the immune response in guinea pigs (35, 36, 37). It has been shown that C2-deficient guinea pigs differ from C4-deficient guinea pigs in that they do respond with an adequate IgG response to high antigenic loads of T-dependent Ags (36). The presence of the C2-bypass pathway may explain these differences between C4-deficient and C2-deficient animals.
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2 Address correspondence and reprint requests to Dr. Michael M. Frank, Department of Pediatrics, Box 3352, Duke University Medical Center, Durham, NC 27710. E-mail address:
3 Abbreviations used in this paper: VBS, Veronal-buffered saline; GVBS2+, VBS with 0.1% gelatin, 0.15 mM CaCl2, and 1 mM MgCl2; EDTA-GVBS, VBS with 0.1% gelatin and 10 mM EDTA; EGTA-GVBS, GVBS2+ with 8 mM EGTA and 5 mM MgCl2; CR1, complement receptor type 1; sCR1, soluble form of CR1 lacking the transmembrane and cytoplasmic domains; sCR1[desLHR-A], sCR1 lacking the first long homologous repeat (LHR-A); CVF, cobra venom factor; MBL, mannan-binding lectin.
Received for publication May 11, 1999. Accepted for publication July 8, 1999.
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), 1:9 (•), and 1:18 (
).
