Hereditary properdin deficiency is linked to susceptibility to meningococcal disease (Neisseria meningitidis serotypes Y and W-135) with high mortality. Its relative contribution toward the outcome of nonseptic shock has not been investigated. Using properdin-deficient C57BL/6 mice and their littermates, this study examines their survival of zymosan-induced and LPS-induced shock. Properdin-deficient mice were more resistant to zymosan shock compared with wild-type mice, which showed greater impairment of end-organ function 24 h after zymosan injection, higher TNF-α production by alveolar and peritoneal macrophages, higher TNF-α, and, inversely, lower IL-10 levels in peritoneal lavage and circulation and higher plasma C5a levels. Properdin-deficient mice showed significantly higher mortality in LPS shock, elevated TNF-α, and, inversely, reduced IL-10 production by peritoneal macrophages as well as lower plasma C5a levels compared with wild-type littermates. NO production by peritoneal macrophages and plasma α1-antitrypsin levels at 24 h after the injection of LPS or zymosan were decreased in properdin-deficient mice in both models, and fewer histopathologic changes in liver were observed in properdin-deficient animals. This study provides evidence that properdin deficiency attenuates zymosan-induced shock and exacerbates LPS-induced shock.

Complement acts in the first line of the immune defense before the generation of a specific, adaptive immune response because it recognizes and subsequently is activated by pathogen-associated molecular patterns and immune complexes involving preimmune Abs of the IgM type. These patterns can be heterogenous and activate one or the other or all three of the distinct complement activation pathways, namely the classical, lectin, and alternative pathways. LPS, a wall component endotoxin of Gram-negative bacteria, activates the classical and lectin pathways of complement (1). Zymosan, a component of yeast cells, initiates the alternative and classical pathways of complement (2, 3). C3- and C4-deficient mice are significantly impaired in their clearance of LPS and show greater mortality in LPS-induced shock (4), mirroring the significance of intact complement activation pathways for the survival of this particular type of nonseptic shock. By contrast, C5-deficient mice show decreased mortality in zymosan-induced shock (5), suggesting that impairment of complement activation involving the generation of C5a, an anaphylatoxin, and C5b, the initiating component of the membrane attack complex, could be beneficial for the survival of this type of nonseptic shock.

In previous work, we have shown that an inhibitor of classical and alternative pathways of complement (fangchinoline) improves the outcome of zymosan-induced nonseptic multiorgan dysfunction syndrome (6, 7, 8). We have further characterized a properdin-deficient mouse line in a model of cecal ligation and puncture that leads to subacute polymicrobial sepsis and found that over an observation period of 14 days, properdin-deficient mice were significantly impaired in their survival compared with wild-type (WT)4 littermates (9). Properdin participates in alternative pathway activation by stabilizing C3 and C5 convertases, thereby amplifying ongoing complement activation initiated by any of the pathways. Recently, the generation of another genetically engineered properdin-deficient mouse line (on mixed background 129/C57BL/6) was reported. In vitro analysis of this properdin-deficient serum using LPS revealed a deficiency in alternative pathway activation, whereas alternative pathway activation induced by zymosan was only marginally impaired and classical pathway-triggered alternative pathway amplification remained intact (10).

The purpose of the present study was to examine, in vivo, the course of zymosan- and LPS-induced models of shock in WT and properdin-deficient C57BL/6 mice.

Experiments were performed using properdin-deficient mice (9) that were backcrossed for 9–12 generations onto C57BL/6 background and WT littermates (all males) weighing 30–35 g. The animals had free access to water and standard chow. The study protocols were approved by the Ethical Animal Commission of the Institute of Microbiology, Sofia, Bulgaria. These properdin-deficient mice were generated by gene-specific targeting and have recently been characterized as being completely impaired in properdin-dependent rabbit erythrocyte lysis compared with their WT littermates; however, they do not differ in their serum levels of C3 and IgM (9).

Zymosan (1 mg/kg body weight; Sigma-Aldrich) was suspended in sterile water and autoclaved for 30 min. WT and properdin-deficient mice were injected i.p. with 1 or 0.8 (low dose) mg/g body weight of this suspension (0.5 ml) or with 400 μg of LPS (Escherichia coli serotype 055:B5, Sigma-Aldrich) per mouse, a dose that elicits severe shock in mice of this genetic background (11), and tested in a pilot experiment to achieve such phenotype in C57BL/6 control mice. Mice injected i.p. with 0.5 ml of saline served as controls.

Mice were bled at various time points, culled, and liver, spleen, and kidneys were removed and weighed. To correct for differences in body weight between animals, organ weights were calculated as a percentage of total body weight.

Livers were fixed in formaldehyde and paraffin embedded. Sections (7 μm) were deparaffinized with xylene and stained with H&E. TUNEL analysis to detect apoptotic cells was performed using TdT-FragEL DNA fragmentation detection kit following the manufacturer’s (Merck Chemicals) instructions and evaluated blind by two assessors.

Blood was collected in a microcapillary pipette (10 cm × 1 mm) containing a horse hair of 20 cm in length as described (12). Every 30 s, 5 mm of the hair was pulled out of the tube manually and formation of a clot was recorded visually.

At day 7 of zymosan inflammation heparinized blood samples were collected and plasma was separated. All samples were analyzed immediately by standard laboratory kits (Dialab), measuring the following biochemical markers of organ dysfunction: alanine aminotransferase (a specific marker for hepatic parenchymal injury), aspartate aminotransferase (a nonspecific marker for hepatic injury), bilirubin (a predictor of liver failure), creatinine (an indicator of reduced glomerular filtration ability), and glucose level.

Peritoneal macrophages (pMφ) were harvested by rinsing the peritoneal cavity with 10 ml of RPMI 1640 medium (Cambrex). The cells were washed twice, resuspended at a density of 2 × 106 cells/ml in RPMI 1640 medium supplemented with 5% (v/v) FCS (Sigma-Aldrich), penicillin (100 IU/ml), and streptomycin (100 μg/ml) (Sigma-Aldrich). A volume of 0.5 ml was dispensed in 24-well plates (Becton Dickinson), and after 1 h at 37°C nonadherent cells were removed by two washings and adherent cells were cultured at 37°C in the presence or absence of 100 μg/ml zymosan or 100 ng/ml LPS. Alveolar macrophages (aMφ) were obtained by repeated bronchopulmonary lavage with 0.6 ml of sterile PBS (pH 7.2). Macrophages were enriched by adherence to plastic (37°C for 1 h) in the presence of 5% (v/v) FCS. The adherent cell concentration and viability were determined by trypan blue exclusion. TNF-α and IL-10 were determined after 18 h of cultivation by quantitative ELISA (Cytolab). The detection limits were <50 pg/ml for TNF-α and 20 pg/ml for IL-10.

Plasma levels of α1-antitrypsin were determined by ELISA as recently described (13).

Peritoneal macrophages were harvested as described above. The cells were washed once and plated at a density of 1 × 106 cells/ml in 200 μl of RPMI 1640 medium for 1 h at 37°C. Nonadherent cells were removed by two washings and adherent cells were cultured at 37°C for 18 h in the presence or absence of LPS (100 ng/ml). Concentration of the stable NO metabolite nitrite (a measure of NO activity) was assayed in culture supernatants by a standard Griess reaction as recently described (13).

Plates (BD Biosciences) were coated with rat anti-mouse C5a (BD Biosciences catalog no. 558027) overnight at room temperature and blocked with 2% BSA for 1 h at room temperature. Plasma samples (diluted 1/5) and serial dilutions of purified C5a (BD Biosciences catalog no. 622597) were added in parallel and incubated for 2 h at room temperature. After washing with PBS and 0.05% (v/v) Tween 20, biotinylated rat anti-mouse C5a (BD Biosciences catalog no. 558028) was added and detected using avidin-peroxidase (1/1000; Peprotech).

Cryostat sections (7 μm) were fixed in formal saline (10%), washed, and, after application of SeroBlock FcR (BUF041A; Serotec), incubated with rabbit anti-mouse C5aR (1/400; BD Biosciences catalog no. 552837) and goat-anti-rabbit IgG, F(ab′)2-tetramethylrhodamine isothiocyanate (1/400; Santa Cruz Biotechnology catalog no. sc-3841). Fluorescence signals were analyzed using Nikon TE300 widefield epifluorescence microscope.

Differences between groups were compared by two-way ANOVA and unpaired t test. Data are presented as mean ± SD. A value of p < 0.05 was considered significant.

Intraperitoneal injection of LPS lead to signs of systemic reaction (dyspnea, diarrhea, and conjunctivitis) and a moderate reduction of body weight (by ∼15%) over the 7-day observation period for both properdin-deficient and WT mice. Eight of 10 WT (80%) but only three of 11 (33%) properdin-deficient animals survived (Fig. 1).

FIGURE 1.

Properdin-deficient mice are significantly impaired in their survival of acute LPS-induced shock. Properdin-deficient mice (n = 11) and WT littermates (n = 10) were injected with LPS (400 μg i.p.) and mortality was recorded over seven days. ***, p < 0.001; two-way ANOVA.

FIGURE 1.

Properdin-deficient mice are significantly impaired in their survival of acute LPS-induced shock. Properdin-deficient mice (n = 11) and WT littermates (n = 10) were injected with LPS (400 μg i.p.) and mortality was recorded over seven days. ***, p < 0.001; two-way ANOVA.

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Histopathologic examination of livers showed periportal and sinusoidal infiltration of inflammatory cells at 24 h and 7 days for WT and properdin-deficient mice. Both showed single cell necrosis at 24 h. Intraparenchymal, granuloma-like accumulations of mononuclear and lymphocytic cells were seen for WT only. WT mice showed more focal hepatocellular necrosis and ballooning degeneration at 7 days than properdin-deficient mice (Fig. 2).

FIGURE 2.

Properdin-deficient and WT mice differ in their liver histology at day 7 after LPS-injection. Liver sections were stained with H&E (original magnification ×400), and are representative for three mice of each genotype.

FIGURE 2.

Properdin-deficient and WT mice differ in their liver histology at day 7 after LPS-injection. Liver sections were stained with H&E (original magnification ×400), and are representative for three mice of each genotype.

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Peritoneal NO was significantly lower in properdin-deficient mice than in WT mice 24 h after LPS injection (Fig. 3,A). At this time point, peritoneal macrophages were purified and restimulated with LPS. Peritoneal macrophages from both groups increased their NO production after LPS treatment, but those from properdin-deficient mice had significantly reduced NO production compared with WT (Fig. 3,B). Peritoneal macrophages isolated from untreated properdin-deficient and WT mice and subsequently stimulated with LPS showed a significant increase in NO production for both groups but no difference between properdin-deficient mice and WT (Fig. 3 C), demonstrating that LPS-induced NO production per se was not defective in properdin-deficient pMφ.

FIGURE 3.

Properdin-deficient mice have reduced nitric oxide production in a model of LPS-induced shock. A, NO was determined in the peritoneal lavage of control mice and 24 h after LPS-injection. B and C, Peritoneal macrophages were harvested 24 h after the administration of LPS and from untreated mice. Cells (1 × 106/ml) were stimulated in vitro with LPS (100 ng/ml) and supernatants were collected after 18 h and assayed for NO. *, p < 0.05; ***, p < 0.001; t test.

FIGURE 3.

Properdin-deficient mice have reduced nitric oxide production in a model of LPS-induced shock. A, NO was determined in the peritoneal lavage of control mice and 24 h after LPS-injection. B and C, Peritoneal macrophages were harvested 24 h after the administration of LPS and from untreated mice. Cells (1 × 106/ml) were stimulated in vitro with LPS (100 ng/ml) and supernatants were collected after 18 h and assayed for NO. *, p < 0.05; ***, p < 0.001; t test.

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At the onset of inflammation (4 h), properdin-deficient mice had higher TNF-α in plasma and peritoneum than WT mice and their coagulation time was significantly shortened compared with WT (data not shown). Alveolar and pMφ were obtained from LPS-injected mice at this time point and restimulated with LPS in vitro. Alveolar macrophages of properdin-deficient mice were impaired in their responses to up-regulate TNF-α production, while pMφ of properdin-deficient mice significantly increased their production on LPS-stimulation (Fig. 4,A). With respect to IL-10, only aMφ of WT mice responded to restimulation, whereas pMφ from properdin-deficient mice showed inhibition in response to LPS stimulation (Fig. 4 B).

FIGURE 4.

Properdin-deficient and WT mice differ in their ex vivo macrophage activation after LPS injection. Alveolar and peritoneal macrophages were obtained from properdin-deficient and WT mice (n = 4 each) 4 h after LPS injection and were cultivated in the presence of 100 ng/ml LPS. Supernatants were collected after 18 h and TNF-α (A) and IL-10 (B) production was measured. Data are expressed as percentages from unstimulated cultures (set at 100%). ***, p < 0.001; t test.

FIGURE 4.

Properdin-deficient and WT mice differ in their ex vivo macrophage activation after LPS injection. Alveolar and peritoneal macrophages were obtained from properdin-deficient and WT mice (n = 4 each) 4 h after LPS injection and were cultivated in the presence of 100 ng/ml LPS. Supernatants were collected after 18 h and TNF-α (A) and IL-10 (B) production was measured. Data are expressed as percentages from unstimulated cultures (set at 100%). ***, p < 0.001; t test.

Close modal

Properdin-deficient mice had significantly lower plasma C5a levels 24 h after LPS-injection compared with WT (Table I). At this time point, properdin-deficient mice had half the number of peritoneal macrophages than WT (2.1 × 106/ml ± 0.4 vs 4.0 × 106/ml ± 0.5), whereas numbers were comparable in the unstimulated state (4.8 × 105 cells/ml vs 4.5 × 105 cells/ml). The concentration of C5a in the peritoneal lavage of LPS-stimulated, properdin-deficient mice at 24 h did not differ significantly from that of control properdin-deficient mice. However, the increase was significant for LPS-stimulated WT compared with control WT mice.

Table I.

C5a concentrations in plasma and peritoneal lavage in WT and properdin-deficient mice 24 h after LPS injectiona

Plasma (ng/ml)Peritoneal Lavage (ng/ml)
WTProperdin DeficientWTProperdin Deficient
Control (n = 4) 15.0 ± 1.2 18.5 ± 2.0 2.5 ± 0.2 2.8 ± 0.3 
LPS injected (n = 4) 102.0 ± 4.0c 69.0 ± 3.9bc 3.8 ± 0.4c 3.1 ± 0.2 
Plasma (ng/ml)Peritoneal Lavage (ng/ml)
WTProperdin DeficientWTProperdin Deficient
Control (n = 4) 15.0 ± 1.2 18.5 ± 2.0 2.5 ± 0.2 2.8 ± 0.3 
LPS injected (n = 4) 102.0 ± 4.0c 69.0 ± 3.9bc 3.8 ± 0.4c 3.1 ± 0.2 
a

Data are expressed as means ± SD;

b

p < 0.01, WT vs properdin-deficient mice;

c

p < 0.001, control vs LPS-injected mice; t test.

Over the 7 day observation period, only two of 10 WT mice (20%) but 5 of eleven properdin-deficient mice (45%) survived zymosan inflammation (Fig. 5). Zymosan-treated WT and properdin-deficient mice exhibited all signs of organ dysfunction 24 h after shock induction. The extent to which the level of glucose in plasma was decreased and the level of creatinine in plasma was increased was higher in WT than in properdin-deficient mice (Table II). Along with greater increases of alanine aminotransferase, aspartate aminotransferase, and bilirubin in WT compared with properdin-deficient mice, this is in keeping with the outcome of the two groups in this model. There was a progressive and significant reduction of body weight in properdin-deficient and WT mice compared with untreated controls (by 18–30%; n = 4). A significant increase of relative spleen and liver weights was observed in both groups during zymosan inflammation, with WT showing a greater increase at day 7 of zymosan inflammation than properdin-deficient mice compared with controls (by 40%).

FIGURE 5.

More properdin-deficient mice than WT mice survive in a model of zymosan induced-inflammation. Properdin-deficient mice (n = 11) and WT littermates (n = 10) were injected with zymosan (1 mg/kg body weight i.p.) and mortality was recorded over 7 days.

FIGURE 5.

More properdin-deficient mice than WT mice survive in a model of zymosan induced-inflammation. Properdin-deficient mice (n = 11) and WT littermates (n = 10) were injected with zymosan (1 mg/kg body weight i.p.) and mortality was recorded over 7 days.

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Table II.

Plasma concentrations of glucose, alanine aminotransferase (ALT), aspartate aminotransferase (AST), bilirubin, and creatinine in WT and properdin-deficient mice 24 h after zymosan injectiona

Glucose (mg/dl)ALT (U/L)AST (U/L)Bilirubin (mg/dl)Creatinine (mg/dl)
UntreatedZymosan TreatedUntreatedZymosan TreatedUntreatedZymosan TreatedUntreatedZymosan TreatedUntreatedZymosan Treated
WT (n = 4) 147.2 ± 5.1 57.8 ± 4.2*** 44.7 ± 3.0 118.2 ± 5.4*** 0.32 ± 0.03 0.64 ± 0.08*** 65.4 ± 4.8 260.2 ± 7.6*** 0.35 ± 0.04 3.64 ± 0.08*** 
Properdin deficient (n = 4) 137.7 ± 6.0 95.0 ± 5.4***,*** 37.7 ± 4.1 63.3 ± 3.5***,*** 0.44 ± 0.02 0.54 ± 0.04 56.2 ± 5.4 112.6 ± 6.5***,*** 0.48 ± 0.05 1.54 ± 0.03***,*** 
Glucose (mg/dl)ALT (U/L)AST (U/L)Bilirubin (mg/dl)Creatinine (mg/dl)
UntreatedZymosan TreatedUntreatedZymosan TreatedUntreatedZymosan TreatedUntreatedZymosan TreatedUntreatedZymosan Treated
WT (n = 4) 147.2 ± 5.1 57.8 ± 4.2*** 44.7 ± 3.0 118.2 ± 5.4*** 0.32 ± 0.03 0.64 ± 0.08*** 65.4 ± 4.8 260.2 ± 7.6*** 0.35 ± 0.04 3.64 ± 0.08*** 
Properdin deficient (n = 4) 137.7 ± 6.0 95.0 ± 5.4***,*** 37.7 ± 4.1 63.3 ± 3.5***,*** 0.44 ± 0.02 0.54 ± 0.04 56.2 ± 5.4 112.6 ± 6.5***,*** 0.48 ± 0.05 1.54 ± 0.03***,*** 
a

Data are expressed as means ± SD; ∗∗∗, p < 0.001, untreated vs zymosan-treated groups; ∗∗∗, p < 0.001, zymosan-injected WT mice vs zymosan-injected properdin-deficient mice; t test.

Histopathologic examination of WT and properdin-deficient mice livers showed periportal infiltration of inflammatory cells at 24 h and 7 days and sinusoidal infiltration at 4 h and 7 days. WT showed hepatocellular necrosis and ballooning degeneration at 24 h. There was more intraparenchymal lymphocytic infiltration in WT at day 7 compared with properdin-deficient mice (Fig. 6).

FIGURE 6.

Properdin-deficient and WT mice differ in their liver histology of at day 7 after zymosan (Zy) injection. Liver sections were stained with H&E (original magnification ×400) and are representative of three mice for each genotype. Controls are the same as in Fig. 2.

FIGURE 6.

Properdin-deficient and WT mice differ in their liver histology of at day 7 after zymosan (Zy) injection. Liver sections were stained with H&E (original magnification ×400) and are representative of three mice for each genotype. Controls are the same as in Fig. 2.

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Peritoneal NO was significantly lower in properdin-deficient mice than in WT 24 h after zymosan stimulation (Fig. 7,A). Peritoneal macrophages were obtained at this time point. Eighteen hours later, cells from properdin-deficient mice showed a significantly impaired NO secretion compared with WT cells, and this finding was observed after zymosan restimulation (Fig. 7,B). Zymosan treatment of peritoneal macrophages isolated from untreated WT and properdin-deficient mice lead to significant reduction of NO (Fig. 7 C).

FIGURE 7.

Properdin-deficient mice have reduced nitrite oxide production in a model of zymosan-induced shock. A, NO was determined in the peritoneal lavage of control mice and 24 h after zymosan (Zym) injection. B and C, Peritoneal macrophages were harvested 24 h after the administration of zymosan and from untreated mice. Cells (1 × 106/ml) were stimulated in vitro with zymosan (50 μg/ml) and supernatants were collected after 18 h and assayed for NO. *, p < 0.05; ***, p < 0.001; t test.

FIGURE 7.

Properdin-deficient mice have reduced nitrite oxide production in a model of zymosan-induced shock. A, NO was determined in the peritoneal lavage of control mice and 24 h after zymosan (Zym) injection. B and C, Peritoneal macrophages were harvested 24 h after the administration of zymosan and from untreated mice. Cells (1 × 106/ml) were stimulated in vitro with zymosan (50 μg/ml) and supernatants were collected after 18 h and assayed for NO. *, p < 0.05; ***, p < 0.001; t test.

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The initiation of zymosan inflammation was followed by a marked decrease in coagulation time at the 4-h time point in WT mice and remained significantly curtailed compared with the coagulation time recorded for the properdin-deficient mice until day 7 (Fig. 8). Rotary thromboelastography was used to assess, for properdin-deficient and WT mice at t0, the rate of initial fibrin formation, the time taken to reach standard clot firmness, the rate of clot growth, and the clot strength, and did not reveal significant differences (data not shown).

FIGURE 8.

Properdin-deficient and WT mice differ in their coagulation time profile in zymosan-induced inflammation. Mice were injected with zymosan and coagulation time was measured at 0 h (n = 4), 4 h (n = 6), 24 h (n = 4), and 168 h (n = 4). **, p < 0.01; ***, p < 0.001; t test.

FIGURE 8.

Properdin-deficient and WT mice differ in their coagulation time profile in zymosan-induced inflammation. Mice were injected with zymosan and coagulation time was measured at 0 h (n = 4), 4 h (n = 6), 24 h (n = 4), and 168 h (n = 4). **, p < 0.01; ***, p < 0.001; t test.

Close modal

In WT mice there was significantly more TNF-α in plasma and peritoneum at 4 h and significantly less IL-10 at 4 h (plasma and peritoneum) and 24 h (peritoneum) compared with properdin-deficient mice (Fig. 9), consistent with the overall outcome of the two groups.

FIGURE 9.

Properdin-deficient and WT mice differ in their profile for TNF-α and IL-10 in the acute phase of zymosan-induced inflammation. Mice were injected with zymosan and their plasma and peritoneal lavages assessed for TNF-α (A and B) and IL-10 (C and D) at 4 h (n = 10), 24 h (n = 5), and 168 h (n = 2). *, p < 0.05; **, p < 0.01; ***, p < 0.001; t test.

FIGURE 9.

Properdin-deficient and WT mice differ in their profile for TNF-α and IL-10 in the acute phase of zymosan-induced inflammation. Mice were injected with zymosan and their plasma and peritoneal lavages assessed for TNF-α (A and B) and IL-10 (C and D) at 4 h (n = 10), 24 h (n = 5), and 168 h (n = 2). *, p < 0.05; **, p < 0.01; ***, p < 0.001; t test.

Close modal

Alveolar and pMφ macrophages were harvested at different points of zymosan inflammation and cultivated for 18 h in the absence or presence of zymosan. Although aMφ from properdin-deficient and WT mice produced comparable TNF-α levels up to 24 h, there was a significant reduction of TNF-α by properdin-deficient aMφ isolated at 168 h (Fig. 10,A). After in vitro stimulation with zymosan, aMφ of properdin-deficient mice had a significantly lower TNF-α production compared with WT over all time points (Fig. 10,B). Peritoneal macrophages isolated from properdin-deficient mice without (Fig. 10,C) and with (Fig. 10 D) subsequent stimulation with zymosan showed a significant reduction in TNF-α secretion over all time points compared with WT pMφ. With regard to IL-10 production by aMφ and pMφ with or without restimulation with zymosan, there was no significant difference between WT and properdin-deficient mice over time (data not shown).

FIGURE 10.

Properdin-deficient and WT mice differ in their ex vivo macrophage activation after zymosan (Zy) injection. Alveolar and peritoneal macrophages were obtained at different time points (0, 24, and 168 h, n = 4; 4 h, n = 6) and were cultivated in the absence of zymosan (A and C) or with 50 μg/ml zymosan (B and D). Supernatants were collected after 18 h and TNF-α production was measured. **, p < 0.01; ***, p < 0.001; two-way ANOVA.

FIGURE 10.

Properdin-deficient and WT mice differ in their ex vivo macrophage activation after zymosan (Zy) injection. Alveolar and peritoneal macrophages were obtained at different time points (0, 24, and 168 h, n = 4; 4 h, n = 6) and were cultivated in the absence of zymosan (A and C) or with 50 μg/ml zymosan (B and D). Supernatants were collected after 18 h and TNF-α production was measured. **, p < 0.01; ***, p < 0.001; two-way ANOVA.

Close modal

A lower dose of zymosan was used to investigate further the cellular responses in properdin-deficient and WT mice (n = 4 each). At 0.8 mg/g body weight, no mortality was recorded for either group. Peritoneal macrophages from these lower dose zymosan-treated mice were purified on day 7 and analyzed for their NO production. At this time point, pMφ from properdin-deficient mice produced significantly less NO than pMφ from WT mice (0.065 ± 0.005 μg/ml vs 0.173 ± 0.006 μg/ml), consistent with the higher dose model. After restimulation with zymosan for 24 h, however, contrary to the higher dose zymosan treatment, NO production was not increased further (0.083 ± 0.004 μg/ml for properdin-deficient mice and 0.139 ± 0.005 μg/ml for WT), but the difference between WT and properdin-deficient mice remained significantly different. Severity of inflammation and extent of organ damage was greater in WT than in properdin-deficient mice also at this nonlethal dose. Seven days after zymosan-injection, WT and properdin-deficient mice again showed a significant increase in relative spleen and liver weights compared with untreated controls, with zymosan-treated WT mice significantly exceeding zymosan-treated, properdin-deficient mice (data not shown). Liver histology at day 7 of zymosan-induced inflammation showed the qualitative difference observed for the higher dose model in terms of more necrosis and more lymphocytic infiltration in WT mice. The serum creatinine level measured at 24 h was significantly more elevated in WT than in properdin-deficient mice (0.35 ± 0.04 mg/dl creatinine (control WT) and 0.48 ± 0.05 mg/dl (control properdin-deficient) vs 2.04 ± 0.05 mg/dl (zymosan-treated WT, n = 4) and 1.14 ± 0.03 (zymosan-treated properdin-deficient, n = 4), confirming the observation made in the higher dose model. C5a was significantly increased in plasma and peritoneal lavage of WT compared with properdin-deficient mice 7 days after zymosan-injection (Table III). At this time point, WT mice showed significantly fewer pMφ than properdin-deficient mice (control WT, 4.5 × 105 cells/mouse; control properdin-deficient, 4.8 × 105 cells/mouse; zymosan-treated WT, 2.4 × 106 cells/mouse ± 0.6; zymosan-treated properdin-deficient, 4.2 × 106 cells/mouse ± 0.5; 2–4 mice each).

Table III.

C5a concentrations in plasma and peritoneal lavage in WT and properdin-deficient mice at day 7 after zymosan injectiona

Plasma (ng/ml)Peritoneal Lavage (ng/ml)
WTProperdin DeficientWTProperdin Deficient
Control (n = 4) 15.0 ± 1.2 18.5 ± 2.0 2.5 ± 0.2 2.8 ± 0.3 
Zymosan injected (n = 4) 51.0 ± 3.0c 29.6 ± 2.5bc 7.8 ± 0.4c 3.5 ± 0.2bc 
Plasma (ng/ml)Peritoneal Lavage (ng/ml)
WTProperdin DeficientWTProperdin Deficient
Control (n = 4) 15.0 ± 1.2 18.5 ± 2.0 2.5 ± 0.2 2.8 ± 0.3 
Zymosan injected (n = 4) 51.0 ± 3.0c 29.6 ± 2.5bc 7.8 ± 0.4c 3.5 ± 0.2bc 
a

Data are expressed as means ± SD;

b

p < 0.01, WT vs properdin-deficient mice;

c

p < 0.001, control vs zymosan-injected mice; t test.

Endotoxin- and zymosan-induced shock are murine models of a nonseptic, systemic inflammatory response syndrome with high mortality in man. The present study shows the significance of properdin in the survival of LPS-induced shock and reveals that properdin has a contributory role in organ damage during zymosan-induced inflammation.

Properdin is able to bind directly to LPS (E. coli 026:B6, Ref. 10 ; and E. coli 055:B5, the serotype used for the in vivo model in this study; data not shown). The lack of properdin resulted in significantly elevated mortality in LPS-induced shock and is likely to reflect the contribution of the alternative pathway amplification loop toward classical and lectin pathway activation triggered through the binding of C1 and mannan-binding lectin to LPS. This extends the findings of a previous study, which concluded that the outcome of LPS-induced shock is dependent on IgM, C3, and C4 (14). LPS, a TLR4 agonist, activates macrophages, resulting in the release of proinflammatory cytokines such as TNF-α, IL-1, IL-6, and IFN-γ. Tissue injury in endotoxemia depends not only on proinflammatory cytokine production but also on the release of counter-regulatory cytokines such as IL-10. LPS induces IL-10 production by macrophages and, in septic patients, its action correlates with increased survival (15). Unexpectedly, properdin-deficient and WT mice did not differ significantly in their plasma TNF-α profile. This may be explained by an acute bone marrow-suppressive effect exerted by doses of LPS lower than the one used in this model (16). Properdin-deficient and WT mice compare in their differential blood leukocyte counts at t0 (data not shown), but a differential susceptibility to LPS-induced bone marrow suppression cannot be excluded. In support of this possibility, we find that the pMφ count at 24 h after LPS injection was significantly less in properdin-deficient mice compared with WT and that properdin-deficient mice did not increase their peritoneal C5a as much as WT mice at this time point compared with unstimulated control mice. WT macrophages produced more NO at 24 h and had greater liver pathology at the 24-h and 7-day time points compared with properdin-deficient mice, consistent with the understanding that overproduction of NO contributes to superoxide-mediated liver injury in the LPS model (17). Properdin-deficient mice were severely compromised in their survival of LPS-induced shock and did not mount a plasma C5a response as the WT mice did. Further studies are needed to investigate the possibility that high levels of C5a in WT mice are able to suppress TLR4-mediated responses (18).

Zymosan-induced inflammation differs from acute endotoxemic shock in that it causes multiple organ injury. Properdin appeared detrimental in the course of zymosan inflammation based on increased NO in peritoneal exudate, NO production by pMφ, relative spleen and liver weights, and plasma parameters (hypoglycemia, plasma alanine aminotransferase, aspartate aminotransferase, bilirubin, and creatinine) of WT compared with properdin-deficient mice. Properdin apparently is involved in the pathogenesis of acute liver dysfunction and renal failure. In terms of mortality, however, additional determining factors seem to be necessary because survival for properdin-deficient mice was only marginally improved compared with that of WT mice. The relative abundance of TUNEL-positive cells was assessed and found not to differ between WT and properdin-deficient mice (data not shown). TNF-α and IL-10 levels are determinants in the outcome of zymosan inflammation (19, 20). In vitro, mainly zymosan-bound, activated complement, leads to macrophage activation and TNF-α release, while uncoated zymosan particles have little effect (21). In vivo, TLR2- and complement receptor-dependence of effector mechanisms are thought to be time dependent (22). The more severe course of zymosan inflammation observed for WT mice, compared with properdin-deficient mice, correlated with the elevated TNF-α level in peritoneal lavages and circulation and simultaneously with a lower IL-10 level in WT at the early phase of developing multiorgan dysfunction. In general, in zymosan-induced inflammation properdin-deficient aMφ and pMφ released less TNF-α spontaneously and after zymosan restimulation but were able to up-regulate their TNF-α production on subsequent LPS stimulation (data not shown). A significant decrease in coagulation time was observed at 4, 24, and 168 h in WT mice. TNF-α, elevated in WT mice, has been described as a procoagulant (23). Furthermore, α1-antitrypsin, which was significantly more elevated in WT at 24 h and day 7 compared with properdin-deficient mice (data not shown), has procoagulant activity (24). The same argument holds true for C5a (25), which was found more elevated in WT mice at day 7. Therefore, increased coagulation may be a reflection of changes in other parameters and not necessarily an expression of the beginning of disseminated intravascular coagulopathy. Additional experiments are needed to investigate the contribution of properdin to zymosan-initiated, alternative pathway convertase-mediated prothrombin cleavage (26).

Systemic inflammation and organ injury were present even after a nonlethal dose of zymosan, but properdin deficiency attenuated this effect. WT mice had significantly elevated C5a levels in plasma as well as peritoneal lavage. At day 7, WT showed greater hepatic C5aR expression than properdin-deficient mice (by immunofluorescence; data not shown). A detrimental role for C5a in zymosan-induced inflammation has been demonstrated (27, 28). Our study of properdin-deficient mice compared with littermate WT mice suggests that pathology in zymosan inflammation is mediated by alternative pathway-dependent C3 as well as by C5 split product generation. Given that the number of pMφ is lower in WT, further studies are needed to clarify whether their recruitment is C5aR independent (22).

In conclusion, this study demonstrates for the first time that properdin deficiency attenuates the symptoms of zymosan-induced inflammation and exacerbates LPS-induced shock.

We thank Dr. Mariya Hristova (University College London) for helpful discussions, the Transgenic Unit team and Biomedical Services of the University of Leicester for excellent maintenance of the mouse colony, Aline Dupont and Dr. Chris Jones (University of Leicester) for thromboelastography analyses, and Irina Elliott for TUNEL analysis.

The authors have no financial conflict of interest.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1

This study was supported by Medical Research Council Grant G0400300 (to C.M.S.).

4

Abbreviations used in this paper: WT, wild type; aMφ, alveolar macrophage; pMφ, peritoneal macrophage.

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