Abstract
Complement C2 receptor inhibitor trispanning (CRIT) is a Schistosoma protein that binds the human complement protein, C2. We recently showed that peptides based on the ligand binding region of CRIT inhibit the classical pathway (CP) of complement activation in human serum, using hemolytic assays and so speculated that on the parasite surface CRIT has the function of evading human complement. We now show that in vitro the C2-binding 11-aa C terminus of the first extracellular domain of CRIT, a 1.3-kDa peptide termed CRIT-H17, inhibits CP activation in a species-specific manner, inhibiting mouse and rat complement but not that from guinea pig. Hitherto, the ability of CRIT to regulate complement in vivo has not been assessed. In this study we show that by inhibiting the CP, CRIT-H17 is able to reduce immune complex-mediated inflammation (dermal reversed passive Arthus reaction) in BALB/c mice. Upon intradermal injection of CRIT-H17, and similarly with recombinant soluble complement receptor type 1, there was a 41% reduction in edema and hemorrhage, a 72% reduction in neutrophil influx, and a reduced C3 deposition. Furthermore, when H17 was administered i.v. at a 1 mg/kg dose, inflammation was reduced by 31%. We propose that CRIT-H17 is a potential therapeutic agent against CP complement-mediated inflammatory tissue destruction.
Complement is an essential part of the early innate immune defense against infectious organisms and for the elimination of immune complexes (ICs).3 Activation of the complement cascade and consequent formation of the C5b-9 membrane attack complex results in lysis of the infectious agent. The proinflammatory response of complement is caused by the release of the anaphylatoxins, C3a and C5a, which indirectly increase both vascular permeability (by mast cell (MC) degranulation and histamine release) and smooth muscle contraction. C5a also promotes chemotaxis and activation of neutrophils to the site of tissue injury or microbial entry. In addition, it increases the neutrophil-endothelial cell adhesion and induces the release of IL-1, IL-6, IL-8, and TNF-α from leukocytes. This inflammatory reaction represents a normal response to microbial invasion or host tissue injury.
ICs are usually processed and cleared without undue problems, but if not, excessive or inappropriate activation of the C system may result in IC disease. Manifested by inflammatory tissue damage, such diseases include Goodpasture’s syndrome (1), rheumatoid arthritis (2, 3), or systemic lupus erythematosus (4). The Arthus reaction is a classic model of IC-mediated inflammation and tissue damage of clinical relevance to a wide range of inflammatory diseases. In the classical direct passive Arthus reaction, the passively immunized animal is challenged with intradermally (i.d.) injected Ag. In the reversed passive Arthus reaction (RPAR), the Ag is injected i.v. and forms ICs with the i.d. injected Ab. The resulting complement and neutrophil-dependent injury is characterized by vascular tissue damage, edema, hemorrhage, and neutrophil influx.
Hitherto, the two main components required to initiate a dermal Arthus reaction have been considered to be the MC via FcγRIII (5) and the IC-activated CP of complement via the interaction of C5a with C5aR (6). The loss of FcγRIII results in a 60% reduction in edema formation and neutrophil recruitment compared with wild-type mice, whereas C5aR deficiency results in a 30–50% reduction (7). That complement is required in the Arthus reaction is further supported by a reduction of the associated acute inflammatory events after depletion of complement with cobra venom factor (8). Following recent studies, a third mediator of the dermal Arthus reaction, namely the influx of polymorphonuclear cell (PMN), can be added. A combined deficiency of the cell adhesion molecules L-selectin and ICAM-1 resulted in a 50–60% inhibition of neutrophil infiltration as well as edema and hemorrhage at the site of IC deposition (9). Despite the many different factors involved, the main mediators in IC-mediated inflammatory damage are thus FcγR, CP complement activation and PMN adhesion, and infiltration.
Several approaches have been used to ameliorate C-mediated IC inflammation and tissue damage in various animal models. Among these has been the use of recombinant soluble versions of membrane-bound complement regulatory proteins, such as recombinant soluble complement receptor type 1 (rsCR1). This inhibits both CP and AP activation and has been administered, both systemically and locally, at therapeutically active levels to inhibit the RPAR (10, 11).
The only complement inhibitor specifically targeting the CP that has been used successfully in vivo is C1-inhibitor (C1-INH). It was able to prevent xenograft rejection of a porcine to primate renal xenotransplant (12). C1-INH therapy was also found to be beneficial in feline and porcine models of myocardial ischemia reperfusion (13, 14), although the latter doses of >100 IU/kg had no benefit and induced coagulation disorders (14). CRIT is a complement receptor found on the surface of the Schistosoma parasite (15). We and others have previously shown that the N-terminal, first extracellular domain of CRIT (ed1) is able to bind the human complement component C2 from plasma and also inhibit the CP of complement activation in vitro (16, 17). An 11-mer synthetic peptide based on the C-terminal part of this region (the 11-aa C terminus of ed1 (H17 or CRIT-H17)) is also able to inhibit the CP in vitro (18). Hitherto, the ability of CRIT to regulate complement in vivo has not been assessed. In this study, we have chosen the RPAR as a classic model of complement-mediated inflammation, to test the capacity of H17 to block inflammation.
Materials and Methods
Mice
Pathogen-free female BALB/c mice (∼20 g and 4 wk old) were obtained from the Charles River Breeding Laboratories (Wilmington, MA) and were rested for 1 wk before the start of the experiment. All experiments were conducted according to Italian laws concerning the use of experimental animals.
Reagents
Endotoxin-free rsCR1 was a kind gift from Avant Immunotherapeutics (Needham, MA). Affinity-purified rabbit anti-CRIT-ed1 was described before (15, 18). This and control rabbit IgG (Sigma-Aldrich, St. Louis, MO) were centrifuged (13,000 × g for 5 min) to remove aggregated immunoglobulins before injection. Evans blue dye, cromolyn (disodium cromoglycate), human albumin (HA), and anti-HA were purchased from Sigma-Aldrich. Normal mouse serum was drawn freshly by cardiac puncture and was either allowed to coagulate at 4°C for 1 h, and then used immediately, or was stored at −70°C. Guinea pig and rat sera were obtained from Sigma-Aldrich. For hemolytic assays, SRBC and hemolysin (rabbit anti-SRBC IgM) were obtained from Dade Behring (Marburg, Germany). Peptides H17 (NH2-HEVKIKHFSPY-CO2H) and H17S (NH2-EKFYHIHSKPY-CO2H) were described elsewhere (18). Flag peptide was NH2-DYKDDDK-CO2H.
Cytokine ELISAs
Cytokine concentrations in serum or tissue samples were measured using commercially available solid phase ELISA kits. Serum TNF-α levels were determined using a murine TNF-α ELISA kit according to the protocols of the manufacturer (Genzyme, Cambridge, MA). To obtain local IL-6, tissue extracts were made by macerating on ice using tweezers and then homogenizing in 10 ml/g cold 10-mM potassium phosphate buffer containing 1 mM EDTA (potassium phosphate buffer (PPB), pH 6). Supernatants were then recovered after centrifugation at 21,000 × g for 15 min. IL-6 was measured using a murine IL-6 ELISA kit (Endogen, Cambridge, MA). IL-6 and TNF-α concentrations were calculated from standard curves derived with recombinant murine IL-6 and TNF-α. The limits of detection for IL-6 and TNF-α were 15 and 10 pg/ml, respectively.
RPAR
After treatment with hot water as vasodilator, seven to eight mice per group were injected i.v. without anesthetic in the lateral tail vein with 200 μl of a solution of HA, 10 mg/kg in 1% Evans blue dye. After 5 min, 10 μl of anti-HA (10 mg/kg) was injected i.d. with a 29-gauge needle under anesthesia (diethyl ether) in the auricular pinna at a median point between artery and vein together with test peptides (H17, H17S, or Flag) or 20 μg/site anti-CRIT-ed1. Ag or Ab controls received either HA only, i.v. (and PBS i.d.) or anti-HA only, i.d. (and PBS i.v.), respectively, both HA and anti-HA having been centrifuged previously at 13,000 × g for 5 min to remove any aggregates. Positive control mice were injected with rsCR1 (30 μg/site). As a second positive control, cromolyn (disodium cromoglycate) was used. This was in preference to the decomplementing cobra venom factor because of its reported generation of proinflammatory C3a and C5a anaphylatoxins (19). Cromolyn was administered at 0.3 mg/kg 30 min before the i.v. injection with HA. Three hours after initiation of the RPAR the animals were sacrificed. Whole blood was collected by intracardiac puncture and kept on ice. After clot formation and retraction, serum was collected by centrifugation at 4°C, and samples were stored at −20°C until evaluation of cytokine concentration. After sacrifice, the ears were taken and extracts were made to evaluate local cytokine levels and myeloperoxidase (MPO) activity (see below).
Quantitative measurement of edema and hemorrhage and pathology assessment
Edema was evaluated by weight. Plasma extravasation as a measure of microvascular permeability or hemorrhage was determined by extraction of extravasated Evans blue dye with formamide from injected ears. For this, an 8-mm diameter part of the ear was removed by punch biopsy. After 24 h in 2 ml of formamide, the ear was homogenized and incubated at 60°C for 2 h. After centrifugation (10,000 × g for 15 min), the extract was filtered and A620 was measured. In addition to the i.d. administration of CRIT-H17 (the test compound at two concentrations), it was also given i.v. (0.1 and 1 mg/kg). Lesions were graded based on severity using a four point scale as follows: 1, minimal; 2, mild; 3, moderate; 4, severe.
Histological examination and immunohistochemical staining of lesion
Serial sections (5 μm) of ear tissue, fixed in 10% buffered formalin and paraffin-embedded, were cut from the edge of the lesion to just before the injection site and were stained with H&E for evaluation of neutrophils. Lesions were then examined using microscopy for cellular influx and edema by an observer blinded of the experimental design.
To reveal C3b deposition, immunohistology was also performed on the sections using a goat anti-mouse C3 polyclonal Ab (Cappel; ICN Pharmaceuticals, Costa Mesa, CA) F(ab′)2. Briefly, mouse ears fixed, embedded, and cut as above were deparaffinated and hydrated according to standard protocols. Blocking of endogenous peroxidase was done with 0.3% H2O2 in methanol for 30 min and by washing three times for 5 min in distilled water and for 5 min in PBS. For blocking of nonspecific staining, sections were incubated with normal rabbit serum, excess serum being blotted away. Goat anti-mouse C3 Ab was used at 1/500 for 60 min at room temperature. After washing in PBS, the biotinylated rabbit anti-goat Ab (Vector Laboratories, Burlingame, CA) was added at 1/50 for 30 min. After washing, the sections were incubated with VECTASTAIN Elite ABC reagent (Vector Laboratories) for 30 min. Upon further washing, sections were incubated with freshly prepared 3-amino-9-ethylcarbazole solution (peroxidase substrate) for 15–20 min until a suitable color had developed. Counterstaining was with Mayer’s hematoxylin for 2 min followed by 2 min of rinsing in tap water and 5 min in distilled water. Finally, samples were mounted with DAKO (Glostrup, Denmark) Faramount aqueous mounting medium. All sections were viewed on a Zeiss (Oberkochen, Germany) Axiophot microscope, and images were acquired on a JVC digital camera (Victor, Yokohama, Japan).
Tissue MPO assay
Assays for MPO activity were made from 1 cm2 skin biopsy punches from the injection sites. Tissue extracts were prepared as described above for the procedure to measure local IL-6. To the homogenized tissue in PPB, 0.5% of the detergent exadecyltrimethylammoniumbromide (Sigma-Aldrich) was added to release MPO from the neutrophils. Cells were broken by three cycles of freezing and thawing followed by sonication. The supernatant (100 μl) was mixed with 2.9 ml of PPB containing 0.167 mg/ml O-dianisidine hydrochloride (Sigma-Aldrich) and 0.0005% hydrogen peroxide. A serial dilution of MPO from human PMN (Calbiochem, La Jolla, CA) was used as a standard, and the MPO released was measured at A450.
Hemolytic assays
These were conducted as described before (18) and were used to assess the species specificity of the complement inhibition due to CRIT-H17. Hemolytic assays were performed using mouse, rat, and guinea pig sera. Mouse serum was obtained by cardiac puncture, and clotting was allowed to proceed for 1 h at 4°C to try to retain complement activity. Rat and guinea pig sera were obtained commercially.
Statistical analyses
A two-tailed Student’s t test was used to determine the statistical significance between treatment groups and positive controls after subtraction of mean background negative control values. All values were expressed as the mean ± SEM. The level of statistical significance was taken as p < 0.05 (significant, ∗) and p < 0.01 (highly significant, ∗∗).
Results
Complement inhibition by CRIT-H17 is species specific
To know whether CRIT-H17-mediated complement inhibition was species-specific, hemolytic studies were conducted with different animal sera. We found that CRIT-H17, as compared with the scrambled peptide, H17S, inhibited rat and mouse complement in a dose-dependent manner, maximal inhibition occurring with ∼1 μM. However, CRIT-H17 did not inhibit guinea pig complement (Fig. 1).
Species-specificity of C-inhibiting H17 peptide. The ability of the H17 peptide in a range up to 1 μM to inhibit the CP of complement activation from guinea pig, rat, and mouse was tested in an in vitro hemolytic assay as described in Materials and Methods. Sham peptide H17S was tested with mouse and rat serum, but data with guinea pig serum are not shown. Various dilutions of sera (1/350 guinea pig; 1/175 rat; 1/10 mouse) were used to obtain between 50 and 80% lysis of Ab-sensitized sheep erythrocytes in the absence of H17. Mean results presented are representative of two or three separate experiments.
Species-specificity of C-inhibiting H17 peptide. The ability of the H17 peptide in a range up to 1 μM to inhibit the CP of complement activation from guinea pig, rat, and mouse was tested in an in vitro hemolytic assay as described in Materials and Methods. Sham peptide H17S was tested with mouse and rat serum, but data with guinea pig serum are not shown. Various dilutions of sera (1/350 guinea pig; 1/175 rat; 1/10 mouse) were used to obtain between 50 and 80% lysis of Ab-sensitized sheep erythrocytes in the absence of H17. Mean results presented are representative of two or three separate experiments.
CRIT-H17-mediated attenuation of RPAR in the skin
The RPAR in mice was used to assess the ability of CRIT-H17 to inhibit complement activation in vivo. In mice it has been speculated that the role of complement is dependent on the strain, complement having been shown to play no role in the RPAR in C57BL/6J mice (20). We chose the BALB/c strain because in this strain IC-triggered inflammation is complement dependent, unlike C57BL/6J mice in which macrophage activation via FcγR is the major requirement (21). The dermal RPAR model we used was initiated in the BALB/c mouse ear. That the interaction of Ab and Ag in the dermis was required in this model was shown by the i.v. injection of Ag alone or the i.d. injection of Ab alone, which resulted in negligible edema and hemorrhage. A preliminary experiment conducted with only four animals per group was used to select an effective dose of CRIT-H17. We found that CRIT-H17 injected within the range 0.01–1 μg together with the i.d. administered anti-HA gave the greatest reduction of edema (measured by weight) and plasma extravasation, as a measure of hemorrhage, each as a percentage reduction of values obtained with mice injected with PBS in place of CRIT-H17, at a 1-μg dose (Fig. 2,A). In a second experiment conducted with seven animals per group, we found that a 1-μg dose of CRIT-H17 compared with a PBS injection, reduced edema and plasma extravasation in RPAR mice by significant levels of 45 (Fig. 2,B) and 41% (Fig. 2 C), respectively. The 0.01-μg dose reduced edema and microvascular permeability by only 26 and 21%, respectively. The i.v. administered CRIT-H17 was used at a dose of 0.1 mg/kg and reduced edema by only 18% and microvascular permeability by 23%. Through its inhibitory effects on MC degranulation, the nonsteroidal anti-inflammatory cromolyn is known to inhibit vasopermeability and could account for the reduced vascular permeability and edema measured in our model. Cromolyn effectively reduces type III (passive Arthus) hypersensitivity reactions (22, 23), directly reduces neutrophil extravasation (24), and reduces plasma extravasation in peritoneal inflammation (25).
H17 reduces IC-induced ear inflammation in terms of edema and microvascular permeability. A, The RPAR was allowed to proceed for 3 h after i.d. injection of H17, at different doses. As a measure of inflammation, edema measured by weight and microvascular permeability measured by extravasated Evans blue were determined, and the average percentage reduction, compared with the positive control (RPAR) group treated with PBS, was calculated. B and C, H17 was administered i.d. at a 1-μg dose along with flag peptide (as negative control for H17); or in D and E, H17S (as negative control for H17) and rsCR1 as positive control for H17. α-ed1 was the anti-CRIT extracellular domain 1 Ab, which cross-reacts with the CRIT-like epitope in the C4 β-chain. Cromolyn acted as an alternative positive control for the reduction of inflammation. Negative controls for the RPAR itself were given either Ag alone i.v., but then no Ab i.d., or no i.v. Ag but only Ab i.d. Data are expressed as the mean ± SEM, (n = 7–8 mice), ∗, p < 0.05.
H17 reduces IC-induced ear inflammation in terms of edema and microvascular permeability. A, The RPAR was allowed to proceed for 3 h after i.d. injection of H17, at different doses. As a measure of inflammation, edema measured by weight and microvascular permeability measured by extravasated Evans blue were determined, and the average percentage reduction, compared with the positive control (RPAR) group treated with PBS, was calculated. B and C, H17 was administered i.d. at a 1-μg dose along with flag peptide (as negative control for H17); or in D and E, H17S (as negative control for H17) and rsCR1 as positive control for H17. α-ed1 was the anti-CRIT extracellular domain 1 Ab, which cross-reacts with the CRIT-like epitope in the C4 β-chain. Cromolyn acted as an alternative positive control for the reduction of inflammation. Negative controls for the RPAR itself were given either Ag alone i.v., but then no Ab i.d., or no i.v. Ag but only Ab i.d. Data are expressed as the mean ± SEM, (n = 7–8 mice), ∗, p < 0.05.
In a third experiment we again found decreases of edema (42%) and plasma extravasation (35%) with i.d. CRIT-H17 (1 μg), compared with the PBS-injected control (Fig. 2, D and E, respectively). The negative control peptide this time was the scrambled peptide sequence, H17S. Two positive controls for CRIT-H17 were used. Recombinant sCR1 (rsCR1) does not show species specificity (26) in its inhibition of C and shows efficacy in numerous in vivo models of complement-mediated disease, including the dermal Arthus reaction, which is ameliorated in terms of neutropenia, tissue immunoreactivity to C3b, and MAC formation (10). The second positive control was cromolyn. The monospecific anti-CRIT-ed1, which specifically recognizes human C4 via a linear epitope in the β-chain (18) also inhibited the inflammatory response (27% reductions in both edema and microvascular permeability) but to a lesser extent than CRIT-H17. We previously suggested (18) the linear epitope on the C4 β-chain as a likely point of interaction between C4b and C2 in assembling the CP C3 convertase. The monospecific anti-CRIT-ed1, by binding the C4 β-chain in this region (F222–Y232), could possibly interfere with CP C3 convertase assembly. Presumably this would be manifest, as we observed, by a reduction of the inflammatory response upon IC-mediated CP activation.
CRIT-H17 can inhibit C-mediated IC inflammation in vivo after i.v. administration
We then wanted to see if at a higher i.v. dose of CRIT-H17 than given previously (0.1 mg/kg) a significant reduction in inflammation could be achieved. The small percentage reduction in inflammation was improved with a 1 mg/kg dose i.v. (Fig. 3, A and B) giving significant reductions of edema (30%) and extravasation (32%).
Capacity of H17 to inhibit C-mediated inflammation in mice after i.v. administration. H17 administered i.v. at a 1 mg/kg dose (but not at 0.1 mg/kg) effectively reduces edema (A) and plasma extravasation (B). Data are expressed as the mean ± SEM, (n = 7–8 mice), ∗, p < 0.05 and ∗∗, p < 0.01.
Capacity of H17 to inhibit C-mediated inflammation in mice after i.v. administration. H17 administered i.v. at a 1 mg/kg dose (but not at 0.1 mg/kg) effectively reduces edema (A) and plasma extravasation (B). Data are expressed as the mean ± SEM, (n = 7–8 mice), ∗, p < 0.05 and ∗∗, p < 0.01.
CRIT-H17-mediated inhibition of leukocyte infiltration and of complement activation at local RPAR site
The visible lesions in terms of edema, which developed in the positive control mice (Arthus reaction) were judged to be moderate on the four-point scale of severity (1, minimal; 2, mild; 3, moderate; 4, severe) but were minimal in CRIT-H17-treated animals. Histological examination of H&E-stained tissue sections in the positive control Arthus reaction (Fig. 4,A) compared with the Ab control (Fig. 4,B) or Ag control (data not shown) showed a severe inflammatory response at the site of i.d. challenge. As shown in Fig. 4,A, this was observed as edema causing separation of collagen fibers and was accompanied by an i.d. polymorphonuclear infiltrate. When CRIT-H17 was also injected (Fig. 4,C) and similarly with rsCR1 (Fig. 4,E), edema, although present, was reduced, and there was a much reduced perivascular and i.d. cellular infiltrate. That the IC-mediated inflammation was attenuated by injection of CRIT-H17 was clear by comparison with mice that received H17S (Fig. 4,D) in which there was no amelioration of the Arthus reaction, a severe cellular influx and edema still being maintained. The qualitative results of neutrophil influx were confirmed by measuring MPO activity from extracts of tissue in which the inflammation was induced. Compared with the RPAR group (treated with PBS) and H17S-treated control, CRIT-H17 reduced the MPO activity by 72% and rsCR1 by 38% (Fig. 4,G). The average reduction in MPO activity (as a measure of neutrophil influx) in the group treated with complement inhibiting CRIT-H17 paralleled the average reduction in serum TNF-α (Fig. 4 H) measured in the same animals. Treatment of the RPAR with cromolyn caused a reduction of MPO activity that similarly paralleled reduced serum TNF-α levels (not shown).
Histological examination of tissue sections reveals that H17 reduces the parameters of IC-mediated ear inflammation in mice, including cellular influx. The dermal RPAR was generated in mouse ears by an i.d. injection of anti-HA following an i.v. injection of HA. A, RPAR; B, Ab only control; Remaining groups in which the RPAR was induced were coinjected i.d. with the following test substances: C, H17; D, H17S; E, rsCR1; and F, cromolyn (given i.v. 30 min before the experiment). Edema is visible in the RPAR as separation of collagen fibers and of neutrophil infiltration. Sections shown are representative of observations from four animals per group. Magnification is ×100 throughout. Abbreviations used: h, hair follicle; c, cartilage; m, muscle. G, As a quantitative measure of neutrophil influx, MPO activity was measured from local tissue extracts for each group in the same experiment, and the percent reduction for H17 treatment was compared with the RPAR (PBS injected) positive control. H, Average values of serum TNF and of MPO activity (as a measure of neutrophilia) compared between the RPAR group (PBS treated) and those in which the RPAR was treated with H17. Data are expressed as the mean ± SEM, (n = 7–8 mice), ∗, p < 0.05. Magnification is ×200 (A–C, E and F) or ×400 (D).
Histological examination of tissue sections reveals that H17 reduces the parameters of IC-mediated ear inflammation in mice, including cellular influx. The dermal RPAR was generated in mouse ears by an i.d. injection of anti-HA following an i.v. injection of HA. A, RPAR; B, Ab only control; Remaining groups in which the RPAR was induced were coinjected i.d. with the following test substances: C, H17; D, H17S; E, rsCR1; and F, cromolyn (given i.v. 30 min before the experiment). Edema is visible in the RPAR as separation of collagen fibers and of neutrophil infiltration. Sections shown are representative of observations from four animals per group. Magnification is ×100 throughout. Abbreviations used: h, hair follicle; c, cartilage; m, muscle. G, As a quantitative measure of neutrophil influx, MPO activity was measured from local tissue extracts for each group in the same experiment, and the percent reduction for H17 treatment was compared with the RPAR (PBS injected) positive control. H, Average values of serum TNF and of MPO activity (as a measure of neutrophilia) compared between the RPAR group (PBS treated) and those in which the RPAR was treated with H17. Data are expressed as the mean ± SEM, (n = 7–8 mice), ∗, p < 0.05. Magnification is ×200 (A–C, E and F) or ×400 (D).
Complement C3 deposition
Deposition of ICs resulting in the activation of the CP of C was observed by immunohistological staining for complement C3 (Fig. 5). Sections from reversed passive Arthus mice reacted with anti-C3 showed deposition of C3 in parts of the dermis. When mice similarly treated were injected with CRIT-H17, there was a markedly reduced reactivity with anti-C3, and in the group treated with rsCR1 there was also almost no reactivity observed. In the cromolyn group there was still C3 deposition as a result of complement activation almost undiminished from the PBS-injected (RPAR) group. In the negative control H17S-treated group, C3 deposition was also observed unchanged from the RPAR group. Rabbit anti-goat biotin-conjugated secondary Abs were used as negative controls for the polyclonal goat anti-C3 and were completely nonreactive with any part of the ear sections.
H17 reduces the level of C3 deposition in the dermal RPAR. IC-mediated inflammation was induced in mouse ears: A, RPAR; B, Ab control. Remaining groups in which the RPAR was induced were injected i.d. with: C, H17; D, H17S; E, rsCR1; or F, cromolyn (given i.v. 30 min before the experiment). C3 deposition, indicated in pink, was monitored by histological analysis of sections using an anti-mouse C3 F(ab′)2. Sections shown are representative of observations from four animals per group. Magnification was ×100 throughout.
H17 reduces the level of C3 deposition in the dermal RPAR. IC-mediated inflammation was induced in mouse ears: A, RPAR; B, Ab control. Remaining groups in which the RPAR was induced were injected i.d. with: C, H17; D, H17S; E, rsCR1; or F, cromolyn (given i.v. 30 min before the experiment). C3 deposition, indicated in pink, was monitored by histological analysis of sections using an anti-mouse C3 F(ab′)2. Sections shown are representative of observations from four animals per group. Magnification was ×100 throughout.
Inhibition of IL-6 and TNF-α expression
RPAR mice compared with Ab or Ag controls showed significantly higher levels of dermal IL-6 even after 3 h (Fig. 6 A). In RPAR mice treated with CRIT-H17 (but not H17S), IL-6 was significantly reduced, a smaller reduction occurring in the rsCR1 group. Cromolyn-treated mice also showed significant reductions in local IL-6. Cromolyn is a nonsteroidal anti-inflammatory drug, and as a membrane stabilizer preventing degranulation, cromolyn can inhibit the release of TNF-α (and histamine) from MCs (27). As TNF-α induces expression of IL-6 (28), it may be that in the cromolyn-treated group the inhibition of TNF-α released principally from local MCs accounts for the reduced levels of local IL-6 at sites of IC-mediated inflammation.
Levels of IL-6 and TNF-α at local inflammatory sites and in serum, respectively, following treatment with H17 and amelioration of inflammatory RPAR. Cytokine levels were determined by specific ELISAs 3 h after initiation of the RPAR, both locally (IL6), from ear tissue in which the IC-mediated inflammation was initiated, and systemically (TNF-α). A percentage reduction on treatment with H17 was calculated compared with the RPAR (PBS injected) positive control. Data are expressed as the mean ± SEM, (n = 7–8 mice), ∗, p < 0.05.
Levels of IL-6 and TNF-α at local inflammatory sites and in serum, respectively, following treatment with H17 and amelioration of inflammatory RPAR. Cytokine levels were determined by specific ELISAs 3 h after initiation of the RPAR, both locally (IL6), from ear tissue in which the IC-mediated inflammation was initiated, and systemically (TNF-α). A percentage reduction on treatment with H17 was calculated compared with the RPAR (PBS injected) positive control. Data are expressed as the mean ± SEM, (n = 7–8 mice), ∗, p < 0.05.
The deposition of ICs has recently been described as not only increasing the levels of cytokine expression at the local inflammatory site (5, 29) but also systemically (30). In the peritoneal RPAR in rats (30) for example, serum IL-6 levels peaked at 2 h after initiation but were the same as negative controls by 4 h. However, in RPAR mice we found no significant increase in systemic IL-6 levels even after 3 h, although measurements were not taken at other time points. Three hours after the induction of RPAR in the skin, although local TNF-α levels were at or below minimal detectable levels, there was a significantly increased level of systemic TNF-α. When compared with the RPAR group (treated with PBS), treatment with CRIT-H17 or with rsCR1, but not H17S, reduced the levels of circulating TNF-α significantly (Fig. 6 B). Because of the high variability (large SEM) of readings within the cromolyn treated group, the reduction in serum TNF-α could not be interpreted.
Discussion
This study showed that the synthetic peptide CRIT-H17, based on the 11-aa C-terminal region of Schistosoma CRIT-ed1, can reduce symptoms of the cutaneous RPAR in BALB/c mice. Three hours after IC deposition as a result of administered HA (i.v.) and anti-HA (i.d.), we measured the RPAR as a massive inflammatory response manifested by increases in edema, hemorrhage (microvascular permeability), neutrophil infiltration, C3 deposition, and systemic TNF-α and local IL-6 production. Coinjection of CRIT-H17 at a dose of 1 μg/site (as opposed to control H17S) with anti-HA reduced edema by an average of 44%, microvascular permeability by 38%, and neutrophilia by 72% and also reduced the levels of TNF-α in the serum and of IL-6 released locally at the site of IC-mediated inflammation. These reductions of the inflammatory parameters typical of the RPAR were broadly of the same level as obtained with one of the most potent inhibitors of C-mediated inflammation, rsCR1. Although we did not try lower doses of rsCR1, we found that in this assay 30 μg/site rsCR1 had the same efficacy in mice as did CRIT-H17 at 1 μg/site.
Furthermore, we showed that CRIT-H17 could inhibit CP complement in heterologous sera from different species. As well as CRIT-H17 inhibiting human complement in vitro (18), it also inhibited rat and mouse C and effectively reduced the dermal RPAR in mice. That CRIT-H17 is able to inhibit mouse and rat complement in vitro means that it could be used in certain animal models of disease. It could also prove useful for testing in models of xenotransplantation. On the basis of the CRIT-H17 inhibition of rat complement in vitro, and also on its ability to inhibit the RPAR in mice when given i.v., the rat model of myocardial ischemia reperfusion (31) would seem a suitable in vivo system in which to further test CRIT-H17.
Current complement-inhibiting anti-inflammatory agents such as rsCR1 (11) and C5a-based C5aR antagonists (32) are of large m.w. A smaller size is more desirable pharmacokinetically as well as in terms of metabolic stability, and in this respect the recently described C5aR antagonist AcF-(OpdChaWR) (33) is a more suitable candidate. CRIT-H17 is derived from the complement regulatory protein, CRIT, which protects the Schistosoma parasite from attack by the human CP of complement. Although CRIT-H17 appears to be nontoxic, it might prove to be immunogenic. CRIT-H17 has a 55% identity (73% when considering conserved residues) to the homologous region F222–Y232 in the β-chain of human C4 (18), and Abs raised against CRIT-ed1 (which contains the CRIT-H17 sequence) recognize C4 (18). The problem with using CRIT-H17 as a therapy for complement-mediated inflammatory diseases might be that its similarity with a human molecule could induce an autoimmune response, and so it may prove appropriate to use the homologous sequence from the C4 protein (18).
We are confident that the C3 deposition observed in RPAR mice treated either with PBS or sham peptide, H17S, is not because of complement activation following cell necrosis, itself induced by the mechanical injury of the injection process (34) as all groups were injected i.d. Furthermore, anesthetic, which could also necrose muscle fibers (35) and lead to complement activation and C3 deposition, was not injected in this experiment. The groups injected with Ab alone or Ag alone showed no C3 deposition, and the remaining groups injected simultaneously with CRIT-H17 or with rsCR1 showed markedly reduced C3 deposition. However, cromolyn, despite reducing all the parameters of IC-mediated inflammation, has no effect on IC formation or complement activation itself and hence does not inhibit C3 deposition.
In response to many alarm situations, including those mediated by endotoxin (LPS) or IC, there is CP and AP complement activation (36, 37) with consequent generation of the proinflammatory anaphylatoxic peptides C3a and C5a (38). The anaphylatoxins also regulate inflammatory cytokines. C3a regulates the synthesis of IL-6 and TNF-α from B lymphocytes and monocytes (39, 40); C5a regulates the synthesis of IL-6 and TNF-α from leukocytes (41, 42) and triggers the release of C-X-C chemokines such as macrophage inflammatory protein-2 from MCs.
Up to 5 h after the IC-mediated stimulation of the peritoneal Arthus reaction, TNF-α is released from infiltrating leukocytes (6, 8, 43). In addition to increased peritoneal levels of TNF-α, serum TNF-α levels were surprisingly also raised (30). Likewise, in the dermal Arthus reaction, TNF-α expression at the mRNA level is increased (9). We also found that TNF-α levels correlated with RPAR parameters of inflammation in the dermal Arthus reaction and that these elevated levels were found systemically. Thus, following initiation of the cutaneous Arthus reaction, in agreement with others (30) we also consider TNF-α as a marker of the inflammatory response. From previous studies of both the peritoneal (27, 43) and dermal Arthus reaction (44), it is known that the release (from MCs) of TNF-α, which chemoattracts neutrophils, is C dependent. In accordance, we observed raised levels of TNF-α and MPO in the dermal RPAR and a subsequent reduction of both parameters on inhibition of C for each animal. At the site of IC deposition, increased levels of both TNF-α and IL-6 is well documented. We found this to be the case for local IL-6 levels, although local TNF-α was in general below the level of detection. IL-6 is produced mainly in local tissues. It is then released into the circulation and has both pro- and anti-inflammatory properties, the latter due to an inhibition of TNF-α release (45). Interestingly, IL-6 (and TNF-α) induces synthesis of the complement-inhibiting classical acute phase proteins C1-INH (46) and C4b-binding protein (47) as well as factor I (48). These anti-inflammatory properties may thus serve to limit the C-mediated IC inflammation.
In summary, we have shown that CRIT-H17 has anti-inflammatory activity in vivo in a model of IC disease. In the RPAR we found that it inhibits vascular leakage as well as cellular influx and neutrophilia. In addition, it suppresses the synthesis and release of the proinflammatory cytokines TNF-α and IL-6. We have shown that it can be administered i.v. as well as i.d., and the inhibition of CP-mediated inflammation was comparable with that achieved with rsCR1. The only other specific inhibitor of the CP to show promise in clinical studies is C1-INH. Therefore, we consider the relatively small CRIT-H17, or potentially the homologous C4β222–232, as a suitable future drug candidate for controlling C-meditated inflammatory tissue destruction, which should be tested in various models of IC-mediated inflammatory disease. We are currently testing these peptides in the rat model of myocardial ischemia reperfusion.
Acknowledgements
We are grateful to Prof. A. I. deBeer for his kind support.
Footnotes
This work was supported by grants from the Swiss National Foundation and the Novartis Foundation.
Abbreviations used in this paper: IC, immune complex; CRIT, complement C2 receptor inhibitor trispanning; CP, classical pathway; PMN, polymorphonuclear cell; C1-INH, C1-inhibitor; ed1, first extracellular domain of CRIT; H17 or CRIT-H17, the 11-aa C terminus of ed1; i.d., intradermal; MC, mast cell; PPB, potassium phosphate buffer; RPAR, reversed passive Arthus reaction; rsCR1, recombinant soluble complement receptor type 1; HA, human albumin; MPO, myeloperoxidase.