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Departments of
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Medicine,
Surgery,
Microbiology, and
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Pathology, University of Alabama, Birmingham, AL 35294;
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BioCryst Pharmaceuticals, Birmingham, AL 35244; and
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Biomedical Sciences Research Center "A. Fleming," Vari, Greece
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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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receptor-triggered pathways. For example, treatment
of rats with recombinant soluble human FcRII was shown to lead to a
dose-dependent inhibition of the RPA response (5), and
targeted deletion of the FcR -chain resulted in markedly attenuated
RPA reactions in mice (6, 7). In addition, it was reported
that in mice deficient in complement proteins C3, C4, or C5 the RPA
reaction develops normally (8). Thus, it was proposed that
IC-initiated inflammation in the RPA response relies on activation of
cellular responses triggered by FcRs and does not require complement
activation (8). Subsequent experiments utilizing mice
deficient only in FcRIII indicated that this receptor is involved in
mediation of the RPA reaction (9). However, in contrast to
the studies using mice deficient in the -chain of FcR, experiments
using FcRIII-deficient animals indicated that in addition to
FcR-triggered pathways, complement activation also contributes
significantly to the mediation of the RPA reaction. Höpken et al.
(10) who used mice deficient in the C5a receptor also
demonstrated that complement contributes to the RPA reaction.
Obviously, the exact roles of FcRs and complement in the RPA
reaction remain equivocal. Nevertheless, the combined data suggested to
us that the degree of dependence on complement- and FcR-mediated
pathways in IC-induced injury is species specific. We tested this
hypothesis by directly comparing the RPA reactions elicited in the skin
of normocomplementemic and decomplemented rats, guinea pigs, and mice
and found that the contribution of complement in the early edematous
phase of inflammation indeed exhibits species specificity; in rats and
guinea pigs complement plays a major role, whereas in C57BL/6J mice no
requirement for complement could be demonstrated. In addition, using
the rat model, we show that complement is activated systemically during
the dermal RPA reaction and that treatment with the serine protease
inhibitor BCX 1470 blocks the inflammatory response. This therapeutic
effect of BCX 1470, and perhaps of other similar inhibitors, may prove
useful for the treatment of human diseases where tissue injury is
initiated by IC deposition and is mediated by complement
activation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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All animals were fed and watered ad libitum and maintained according to protocols established by the Animal Resources Program at the University of Alabama at Birmingham. Eight-week-old C57BL/6J mice (The Jackson Laboratory, Bar Harbor, ME) weighing 30 ± 2 g, 12-wk-old Sprague Dawley rats (Charles River Laboratories, Boston, MA) weighing 343 ± 15 g, and 18-wk-old HsdPoc:DH guinea pigs (Harlan Sprague-Dawley, Indianapolis, IN) weighing 278 ± 4 g were used.
Materials
Polyclonal rabbit anti-sheep RBC serum, chicken egg albumin (grade VII), Evans blue dye, 5,5'-dithiobis (2-nitrobenzoic acid), Na-benzoyl-L-Arg-nitroanilide, properdin, and DMSO were from Sigma (St. Louis, MO). Benzyloxycarbonyl-Lys-thiobenzyl was from Nova Biochem (La Jolla, CA), bovine trypsin was from Worthington Biochemical (Freehold, NJ), and human C1s was from Calbiochem (San Diego, CA). Cobra venom factor from Naja naja (CoVF) was from Quidel (San Diego, CA), and sheep and guinea pig erythrocytes were from Colorado Serum (Denver, CO). Polyclonal rabbit anti-chicken egg albumin IgG was from Cappel Laboratories (Cochranville, PA). The human complement proteins C3, factor B, and factor D were purified as detailed elsewhere (11, 12, 13). BCX 1470 (2-amidino-6-[2-thiophene carboxy] benzothiophene methanesulfonate; C14H10N2O2S2) was synthesized by BioCryst Pharmaceuticals (Birmingham, AL) and dissolved in DMSO for in vitro analyses or in 5% dextrose for in vivo use. Complete details of the structure and synthesis of BCX 1470 have been described elsewhere (14).
Determination of the esterolytic activity of factor D, C1s, and trypsin
The esterolytic activity of factor D, C1s, and trypsin was determined by measurement of their ability to hydrolyze appropriate synthetic substrates (15, 16). All esterolytic assays utilized 1.29 mM of substrate dissolved in 0.1 mM HEPES, 0.5 M NaCl, and 10% DMSO and were performed in 96-well microtiter plates (200 µl per reaction). In these assays, hydrolysis of the substrate benzyloxycarbonyl-Lys-thiobenzyl by factor D (104 nM) or by C1s (3–13 nM) liberates an active group which reacts with the chromogen 5,5'-dithiobis (2-nitrobenzoic acid), whereas trypsin hydrolyzes the chromogenic substrate Na-benzoyl-L-Arg-nitroanalide. In both cases the change in absorbance of the colored end-product is monitored at 405 nm for 200 s using a Vmax Kinetic Microplate Reader (Molecular Devices, Menlo Park, CA), which automatically calculates the rate of substrate conversion (mOD/min). To measure the ability of BCX 1470 to inhibit the esterolytic activity of factor D, C1s, and trypsin, the inhibitor was added to 100 µl of assay buffer already containing the target enzyme. Substrate (100 µl) was then added and its conversion was monitored. Negative control wells received buffer, substrate, and inhibitor but no enzyme. The IC50 value for esterolysis was calculated from semilogarithmic plots of percent inhibition of esterolysis vs concentration of inhibitor, with percent inhibition defined as: [(rate of esterolysis in absence of inhibitor - rate of esterolysis in the presence of inhibitor) ÷ (rate of esterolysis in absence of inhibitor)] x 100.
Hemolytic assays
Total classical pathway hemolytic activity of human and rat serum was measured by mixing 100 µl rabbit Ab-sensitized sheep erythrocytes (1.5 x 108/ml) with 100–600 µl of an appropriate dilution of serum in GVB2+ (veronal-buffered saline (pH 7.3) containing 1 mM MgC12, 0.15 mM CaCl2, and 0.1% gelatin). The reaction volume was adjusted to 1.5 ml with GVB2+ and the mixture was incubated for 60 min at 37°C. The absorbance of the supernatants at 413 nm was then used to calculate CH50 U/ml. Total alternative pathway hemolytic activity of human serum was measured by mixing 250 µl rabbit erythrocytes (1 x 108/ml) with 50–150 µl of a 1/10 dilution of serum in DGBV-Mg-EGTA (half-strength veronal-buffered saline (pH 7.3) containing 2.5% dextrose, 2.5 mM MgC12, 10 mM EGTA, and 0.1% gelatin). DGVB-Mg-EGTA was added to a final volume of 500 µl, and the mixture was incubated for 60 min at 37°C. The hemolytic reaction was stopped by adding 2 ml ice-cold DGVB-EDTA (DGVB containing 10 mM EDTA) and the absorbance of the supernatants at 413 nm was used to calculate AP50 U/ml. To measure alternative pathway activity of rat serum, guinea pig erythrocytes were substituted for rabbit erythrocytes. To assess inhibition of classical or alternative pathway hemolytic activity by BCX 1470, dilutions of the inhibitor in 100 µl buffer were added to titration mixtures before adjusting the final volume. Percent inhibition was calculated from controls without inhibitor and used to determine IC50 values.
Factor D hemolytic activity was measured by using neuraminidase-treated sheep erythrocytes carrying human C3b (EC3b), prepared as described (17). Mixtures of 2.5 x 106 EC3b, 250 ng factor B, 6 ng properdin, and variable amounts (0.1–8.0 ng) of factor D in a total volume of 150 µl DGVB-Mg-EGTA were incubated at 30°C for 15 min to allow formation of EC3bBb(P). The reaction was stopped by adding 1.0 ml GVB-EDTA (veronal-buffered saline (pH 7.3) containing 10 mM EDTA and 0.1% gelatin), and the cells were pelleted and resuspended in 50 µl GVB-EDTA. Convertase sites were developed by adding 350 µl guinea pig serum diluted 1/40 in GVB-EDTA and were incubated for 60 min at 37°C. Factor D hemolytic activity was then calculated (18) in U/ng from the absorbance of the supernatants at 413 nm. To assess inhibition of factor D hemolytic activity by BCX 1470, an amount of factor D corresponding to 1 hemolytic unit (1–2 ng of factor D depending on the batch of EC3b cells) was incubated with variable amounts of the inhibitor at 30°C for 30 min, and residual factor D activity was measured. Percent inhibition was calculated from controls without inhibitor and used to determine IC50 values. To assess inhibition of factor B hemolytic activity, EC3bBb(P) cells carrying an average of one hemolytic site per cell were incubated with variable amounts of BCX 1470 in a final volume of 100 µl GVB-EDTA at 30°C for 10 min. Residual sites were then developed with guinea pig serum diluted 1/40 in GVB-EDTA and used to calculate IC50 values.
Arthus reactions
Animals were anesthetized by i.p. injection of a mixture of ketamine and xylazine (Fort Dodge Laboratories, Fort Dodge, IA), and the medial surface of both hind legs and the back was shaved. Chicken egg albumin (6.6 mg/ml) in sterile 0.9% NaCl containing 2% Evans blue dye was injected into the left femoral vein of each animal (10 mg OVA/kg). Five minutes later, the indicated amounts of rabbit IgG anti-chicken egg albumin in 25 µl of 0.9% NaCl was injected intradermally. Control sites received 25 µl of 0.09% NaCl. Before injection of OVA or Ab, any aggregates were removed by centrifugation at 12,000 x g for 5 min. Each animal’s skin was injected with IgG at two sites and with saline at two sites. All intradermal injections were spaced no more than 2 cm apart and were confined to the upper two-thirds of the trunk. At various times after initiation of the RPA reaction, blood was collected for determination of serum hemolytic activity or the animals were killed by CO2 asphyxiation. The injected area of skin was harvested for quantitation of lesion size and was further processed for histology.
The area of the dermis stained by extravasation of Evans blue dye was used to quantitate the degree of inflammation elicited by the RPA reaction. Immediately after harvest, the skins were everted, overlaid with transparent plastic film, and the perimeter of each blue lesion was traced using an electronic hand-held digitizer that automatically calculates the area (in cm2) of each lesion. The net area of lesions was determined by subtracting the average area of lesions generated by injection of saline from the area of each IgG-injected lesion.
Hypocomplementemia was induced via i.p. injection of CoVF (30 µg) (19) 18 h before initiation of RPA reactions. We have shown (20) using ELISA (21) that this treatment reduces mouse serum antigenic C3 to less than 3% of initial levels within 4 h, and that the hypocomplementemic condition persists for at least 48 h. We verified that after injection of CoVF, antigenic C3 was also not detected in rat and guinea pig sera collected up to 24 h after CoVF treatment (data not shown). To test whether administration of BCX 1470 inhibits the RPA reaction, the compound was mixed with the OVA/dye solution and administered i.v. to rats (10 mg/kg) via the right femoral vein immediately before initiation of dermal RPA reactions. Alternatively, BCX 1470 was infused during the initial hour of the RPA reaction (0.25 mg/kg final dose), beginning immediately after injection of OVA and just before dermal injection of Ab. Preliminary tests confirmed that 24-h survival of rats treated with 10 mg/kg of BCX 1470 was 100%. Control rats (no inhibitor) received a bolus or an infusion of 5% dextrose.
Histology
Skin biopsies were taken from saline-injected and Ab-injected sites, embedded in OCT compound (Sakura Fineteck USA, Torrance, CA), flash frozen in liquid nitrogen, and stored frozen (at -70°C) until processed. Serial sections (4 µm) were cut from each lesion on a cryostat microtome set at -20°C, beginning at the periphery of each lesion and ending proximal to but not including the point of injection. Sections were mounted on glass slides, air dried, acetone fixed, and stained with hematoxylin and eosin to visualize overall tissue pathology. To reveal ICs and the extent of deposition of C3, sections were blocked with normal horse serum and double stained with tetramethyl rhodamine isothyocyanate (TRITC)-labeled goat F(ab')2 anti-rabbit IgG and FITC-labeled goat anti-rat C3 (ICN Pharmaceuticals, Aurora, OH). To reveal the injected Ag, tissue sections were stained with FITC-labeled rabbit anti-chicken egg albumin (ICN Pharmaceuticals). Sections were washed and mounted in Fluormount G (Southern Biotechnology Associates, Birmingham, AL) and were viewed with a Leica/Leitz DMRB fluorescence microscope equipped with appropriate filter cubes (Chroma Technology, Brattleboro, VT). Images were acquired with a C5810 series digital color camera (Hamamatsu Photonics, Bridgewater, NJ) and processed with Adobe Photoshop and IP LAB Spectrum software (Signal Analytics Software, Vienna, VA). Images of TRITC fluorescence (red), when superimposed with images of FITC fluorescence (green), produced a yellow-orange color where TRITC and FITC coincided.
Statistical analysis
Significant differences were determined by Student’s t tests. Statistical significance was defined as p < 0.05. All averaged values are expressed as the mean ± SEM.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To determine whether the role of complement in IC-initiated
inflammatory reaction is species specific in rodents and to identify an
appropriate animal model for testing complement inhibitors in vivo, we
compared the RPA reaction in the skin of rats, guinea pigs, and mice.
Under identical experimental conditions, intradermal injection of IgG
anti-chicken egg albumin (50 µg) into animals receiving i.v. 10
mg/kg of chicken egg albumin elicited inflammatory skin lesions in all
three rodent species (Fig. 1
). As judged
by similar lesion size, the inflammatory response was of comparable
intensity in rats and mice but was much more pronounced in guinea pigs
(Fig. 1). The intensity of the RPA response was reduced significantly
(6-fold) in rats made hypocomplementemic by injection of CoVF and was
also reduced significantly (2-fold) in hypocomplementemic guinea pigs
(Fig. 1). In contrast, the size of RPA lesions elicited in mice was not
reduced by CoVF-induced hypocomplementemia (Fig. 1). Thus, in rodents,
sensitivity to RPA-induced extravasation is species specific, and in
rats and guinea pigs, but not in mice, complement appears to be a major
mediator of the inflammatory response.
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Because the net effect of hypocomplementemia on the RPA response
was greatest in the rat, we studied in more detail the kinetics and IgG
dose responsiveness of the reaction in this species. As shown in Fig. 2, dermal lesions are already of
substantial size by 2 h after injection of 20 µg IgG Ab, and
typically they attain maximum size within 3–4 h. Beyond 4 h there
is gradual regression of the size of the lesions, although they are
still obvious even at 18 h. The intensity of the dermal edema
increased with the amount of injected IgG (Fig. 3). A strict requirement for complement
was confirmed by the fact that hypocomplementemic rats showed no
significant edema development even when 100 µg of IgG was used (Fig. 3). The dermal edema elicited by this dose of Ab in hypocomplementemic
rats was not significantly greater than that elicited in control rats
by injection of saline (Fig. 3).
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B), whereas
saline-injected skin showed normal histology (Fig. 4A).
Immunofluoresence confirmed the presence of ICs in Ab-injected sites
(Fig. 5). These contained rabbit IgG
(Fig. 5B), chicken egg albumin (Fig. 5C), and
extensive deposits of C3 (Fig. 5B). By comparison, in the
dermis of hypocomplementemic rats, IgG was deposited (Fig. 6C) but C3 was not (Fig. 6D). These combined data indicate that in the rat during the
first 4 h of the RPA reaction, the dermal inflammatory response
requires the deposition of ICs and an intact serum complement
system.
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The serine protease inhibitor BCX 1470 inhibits the esterolytic
activity of factor D and C1s 3.4- and 200-fold better, respectively,
than that of trypsin (Table I). The
ability of BCX 1470 to inhibit esterolytic activity translates into
potent inhibition of the proteolytic activity of C1s, factor D, and
consequently of the classical and alternative pathway-mediated
hemolysis of target RBC (Table I). As expected from its more effective
inhibition of C1s esterolytic activity, classical pathway hemolytic
activity is inhibited 7-fold more effectively than that of the
alternative pathway. Inhibition of the alternative pathway is
apparently due to inhibition of factor D and not factor B (Table I).
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). Remarkably, the inhibition of edema
development achieved by 1-h infusion of BCX 1470 was equivalent to that
achieved by induction of hypocomplementemia with CoVF (Fig. 7).
Evidence that the mode of action of BCX 1470 in vivo involves
inhibition of complement activity was obtained by serial measurements
of serum hemolytic activity during development of RPA lesions (Fig. 8). During the RPA reaction in control
rats, no significant change in serum classical pathway hemolytic
activity was detected, but alternative pathway hemolytic activity
decreased by 50% (Fig. 8A). By comparison, in rats infused
for 1 h with BCX 1470 the decrease in alternative pathway activity
was delayed by at least 2.5 h (Fig. 8B). Based on the
in vitro data, we suggest that BCX 1470 abbrogates the RPA reaction in
rats via blockade of complement activation, most likely via
inactivation of C1s and factor D enzymatic activity.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Our data support the proposal that complement-mediated processes are of
major importance in the RPA reaction in rats (22). The
weak dermal response we observed in hypocomplementemic rats after
administration of a high dose of Ab and the weak RPA reaction seen by
others in rats treated with the complement inhibitor soluble complement
receptor type I, sCR1 (22), suggest that
complement-independent processes do not make a significant contribution
to the initial increased vascular permeability of the RPA reaction. Our
data show that guinea pigs are more sensitive to IC-mediated
inflammation than rats, but the contribution of complement is likely
similar in these two species. In contrast, complement has little or no
effector role in RPA reaction in C57BL/6J mice. Thus, our data do not
contradict the proposal (23) that in mice, complement is
not required for IC-mediated inflammation. However, because only
C57BL/6J mice were used in our work, we cannot exclude the possibility
of complement participation in the dermal RPA reaction in other
strains of mice. In fact, soon after the initial submission of this
paper, it was reported that the major mechanism promoting immune
complex-triggered peritonitis in C57BL/6J mice was activation of
macrophages via FcRI, whereas in BALB/c mice the response was
complement dependant (24).
The rat RPA reaction has often been used as an in vivo model to test the efficacy of anti-inflammatory drugs (25, 26, 27, 28, 29, 30, 31). Yet, despite these efforts, an extensive literature search suggests that ours is the first study to quantitate the effects of an RPA reaction on the activity of serum complement. Despite evidence that ICs activate the classical pathway in vitro (32), we detected no change in rat serum classical pathway hemolytic titers during the dermal RPA reaction. In contrast, the serum alternative pathway hemolytic activity decreased significantly during the rat RPA response. We do not know the reason for this discrepancy. The results of both hemolytic assays depend on the levels of C3 to C9, although not necessarily to the same extent. Therefore, the observed difference could be due either to a greater degree of activation/consumption of factor B than C2 and/or C4, or to different sensitivities of the two assays to decreased levels of C3 to C9.
We have shown that the synthetic serine protease inhibitor BCX 1470 is a potent inhibitor of C1s and factor D in vitro and that it prevents the initial phase of IC-mediated inflammation in rats and the associated systemic complement activation. We did not exhaustively investigate the effects of the inhibitor on tissue deposition of the various complement components during the Arthus reaction, but presumably deposition of C1q is not affected, whereas deposition of later components (e.g., C3, C4, and C5b-9) is reduced. Also we cannot exclude the possibility that the BCX 1470 effects on RPA were at least in part due to inhibition of some other serine protease. However, taken together with the results of the CoVF decomplementation experiments, these results support the conclusion that complement is necessary for the development of RPA lesions in rat skin. Although the role of complement in IC-mediated diseases may differ in rats and humans, BCX 1470 or other similar protease inhibitors have potential medical applications in the pharmacologic control of complement activation in human diseases.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Alexander J. Szalai, Division of Clinical Immunology and Rheumatology, Department of Medicine, University of Alabama, Birmingham, AL 35294-0006. E-mail address:
3 Current address: Division of Rheumatology, MetroHealth Medical Center, Cleveland, OH 44109.
4 Abbreviations used in this paper: IC, immune complex; RPA, reverse passive Arthus; CoVF, cobra venom factor from Naja naja; EC3b, neuraminidase-treated sheep erythrocytes carrying human C3b; TRITC, tetramethyl rhodamine isothyocyanate.
Received for publication April 20, 1999. Accepted for publication October 13, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RII: production, characterization, and inhibition of the Arthus Reaction. J. Exp. Med. 178:1617. chain deletion results in pleiotrophic effector cell defects. Cell 76:519.[Medline]
RIII (CD16) deficient mice. Immunity 5:181.[Medline]
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) and CoVF-treated
rats (•) using increasing doses of IgG Ab. The area of dermal lesions
was measured at 4 h. The data are presented as the mean ±
SEM from two experiments, n = 2–4 rats (4–8
lesions) per point. *, p < 0.005 vs
CoVF-injected rats receiving an equivalent amount of IgG (Student’s
t test).
