Fungal peritonitis is an important complication in peritoneal dialysis patients; either continuous or recurrent peritonitis may enhance peritoneal damage. Even when the peritoneal dialysis catheter is removed in patients with fungal peritonitis, peritoneal fibrosis can progress and evolve into encapsular peritoneal sclerosis. It is unclear why fungal infections are worse than bacterial in these respects. Zymosan is a cell wall component of yeast that strongly activates the complement system. In this study, we compared the effects of zymosan and bacterial LPS on peritoneal inflammation in a rat peritoneal injury model induced by mechanical scraping. Intraperitoneal administration of zymosan, but not LPS or vehicle, caused markedly enhanced peritonitis with massive infiltration of cells and deposition of complement activation products C3b and membrane attack complex on day 5. In rats administered zymosan and sacrificed on days 18 or 36, peritoneal inflammation persisted with accumulation of ED-1-positive cells, small deposits of C3b and membrane attack complex, exudation of fibrinogen, and capillary proliferation in subperitoneal tissues. When zymosan was administered daily for 5 days after peritoneal scrape, there was even greater peritoneal inflammation with peritoneal thickening, inflammatory cell accumulation, and complement deposition. Inhibition of systemic complement by pretreatment with cobra venom factor or local inhibition by i.p. administration of the recombinant complement regulator Crry-Ig reduced peritoneal inflammation in zymosan-treated rats. Our results show that yeast components augment inflammation in the injured peritoneum by causing complement activation within the peritoneal cavity. Local anticomplement therapy may therefore protect from peritoneal damage during fungal infection of the peritoneum.

Infective peritonitis is one of the most important and serious complications in patients on peritoneal dialysis (PD)3 therapy. Peritoneal dysfunction and sclerosis related to recurrent and/or severe infective peritonitis are risk factors for the future development of encapsulating peritoneal sclerosis (EPS) (1). Fungal peritonitis, caused by yeast infection, most often Candida sp., is not common but carries a poor prognosis (2, 3, 4). In fungal peritonitis, removal of the PD catheter is recommended to rescue the PD patients according to the 2005 guidelines from the International Society for Peritoneal Dialysis (5). However, even a single episode of fungal peritonitis, successfully treated, can induce EPS in the patient (1, 6).

The complement system plays important roles in innate immunity and protection of the host from pathogens. Complement components are mainly produced in the liver and also generated in tissues to play roles in the local area. Mesothelial cells can also produce complement components (7, 8). Activation of the complement system has been shown to protect against microorganisms in the peritoneal cavity (9). However, unregulated complement activation causes tissue damage in many diseases (10, 11) and, as a consequence, anticomplement therapies to control inflammation are now emerging into clinical practice (11). In PD, peritonitis and the subsequent peritoneal sclerosis are associated with inflammation and complement activation. Although PD fluid (PDF) from patients was shown to contain complement components (12), there is no data linking PD peritonitis with complement activation and no in vivo data to implicate complement. Several animal models of PD peritonitis have been developed to investigate peritoneal sclerosis and EPS. Although some showed impressive inflammation, these were rather artificial peritoneal fibrosis models, such as the chlorhexidine gluconate-induced model (13), the long-term acidic PDF exposure model (14, 15), bleach-induced model (16), and silica-induced model (17). Recently, Margetts et al. (18) reported a new peritoneal sclerosis model driven by adenovirus-expressed TGFβ2 (AdTGFβ2), which showed the importance of TGF-β in driving peritoneal inflammation. We recently described a peritoneal injury model induced by controlled peritoneal scraping to induce peritoneal inflammation that was accompanied by increased TGF-β production and evolved into peritoneal fibrosis (19). The peritoneal injury was caused by physical stress and rendered the peritoneum susceptible to the inflammatory trigger. The physical scraping in this model might reflect catheter-related injury and the other physical stresses caused by PDF. These situations might contribute to susceptibility to infection. However, in the absence of the inflammatory drive, the physical injury rapidly resolved. In fact, it should be considered that infectious PD peritonitis in the clinical situations is complexly intertwined with infectious inflammation and other physical stresses. This model appears to follow the natural course of peritoneal fibrosis resembling scar formation, as occurs, for example, after peritoneal damage caused by infection. However, the mechanisms underlying PD-related peritoneal sclerosis and EPS still remain unclear. In particular, it is unclear why fungal peritonitis in PD patients is so severe and so often develops into peritoneal sclerosis and EPS (1, 6). In the various fungal species, the cell wall has numerous biological activities, including activation of the complement system (20). Zymosan is an abundant cell wall component of yeast which activates the complement system through the alternative pathway (21).

In the present study, we examined under conditions of physical stress whether i.p. administration of zymosan replicated the severe peritoneal proliferation seen in fungal peritonitis and whether zymosan as a surrogate for fungal infection were more damaging than LPS as a surrogate for bacterial infection. We also investigated whether complement activation was responsible for the zymosan-related proliferative peritonitis and explored the use of local anticomplement therapy to reduce postfungal peritoneal injuries.

Male Sprague Dawley rats weighing ∼250 g (Chubu Kagaku Shizai) were used in the present study. All experiments described were conducted according to The Animal Experimentation Guide of Nagoya University School of Medicine.

Zymosan A was purchased from Sigma-Aldrich. LPS prepared from phenolic extracts of Salmonella typhimurium and Escherichia coli (055:B5) were obtained from Sigma-Aldrich. Dianeal PD-4 4.25% was used as a 4.25% PDF (pH ∼ 5.0; Baxter).

To investigate expression of rat complement regulators (CRegs), sections were incubated with anti-rat Crry (TLD1C-11; a gift from Prof. W. Hickie, Dartmouth, Hanover, NH) (22), anti-rat DAF (RDIII-7) (23), or anti-rat CD59 (6D1) (24). The bound mAbs were detected using FITC-labeled rabbit anti-mouse IgG (Cappel Labs) absorbed with normal rat serum. To observe C3b and membrane attack complex (MAC; C5b-9) deposition, we used FITC-rabbit anti-rat C3 (Cappel Labs) and polyclonal (pc) rabbit anti-rat C9, respectively (22, 25, 26), followed by incubation with FITC-goat anti-rabbit IgG (Cappel Labs). We also used the anti-human C3b mAb C3/30 that cross-reacts with rat C3b (27).

An Ig fusion protein comprising rat Crry SCR1, 2, 3, and 4 attached to IgG-Fc (Crry-Ig) was prepared as previously described (28, 29, 30). Briefly, cDNA encoding SCR1, 2, 3, and 4 of rat Crry was cloned into the expression vector pDR2ΔEF1α (gift from Dr. I. Anegon, INSERM Unité 437, Nantes, France) upstream of and in-frame with DNA encoding the hinge and Fc domains of rat IgG2a. Vent DNA proofreading polymerase was used in the PCR, and sequencing confirmed that no errors had been introduced by PCR. Chinese hamster ovary cells were transfected with plasmid using Lipofectamine (Life Technologies) according to the manufacturer’s instructions. Stable lines were generated with 400 μg/ml hygromycin B (Life Technologies) in Ham’s F12 medium with 10% FCS and maintained in hygromycin B (100 μg/ml) in RPMI 1640 medium with 5% FCS.

To isolate the fusion protein, spent medium was collected and passed over a TLD1C-11 (anti-Crry) Sepharose affinity column, the column was washed, and Crry-Ig was eluted with 0.1 M glycine-HCl (pH 2.5). The eluted protein was neutralized, dialyzed into PBS, and concentrated by ultrafiltration.

We modified a rat peritoneal injury model induced by mechanical peritoneal damage as described in our previous report (19). Briefly, on day 0, rats were anesthetized, the peritoneal cavity was opened with a mid-line incision, and the right side parietal peritoneum was diffusely scraped with the rim of a sterile 15-ml polypropylene tube (BD Falcon Conical Centrifuge tubes; BD Biosciences) for 60 s. Before undertaking the major experiments, we first examined the effects of different daily doses of zymosan (0.1, 1, and 5 mg/animal) for up to 5 days to define the optimal amount of zymosan. To investigate the in vivo effect of exposure to zymosan on the injured peritoneum, zymosan in PDF was administered i.p. or the vehicle (PDF) alone was administered according to the experimental protocol in Table I. We first examined the effects of zymosan injections given daily with PDF for up to 5 days (group 2) or PDF only (group 1). Animals in groups 1 and 2 were killed on day 1 (n = 6 for each group), day 3 (n = 6), day 5 (n = 6), day 18 (n = 6), and day 36 (n = 6). In a separate group of rats, we gave a single zymosan injection on day 0, followed by four daily vehicle injections (group 3); peritoneal pathology was estimated on day 5 (n = 6). Blood was collected from the tail vein immediately before sacrifice. The parietal peritoneum of the scraped right side was observed macroscopically, then the peritoneal membrane was dissected and divided into four tissue blocks (each 5 mm by 10 mm) for light microscopic (LM) and immunofluorescence analysis. Each i.p. injection was delivered through the unscraped left side.

Table I.

Experimental protocol in the scraping peritonitis model

GroupTreatment (i.p.)Amount (mg/rat/each i.p.)Volume and Timing of i.p. Treatment (ml)
Day 0Day 1Days 2–4
PDFa only — 10 
Zymosan + PDF 5 mg 10 
Zymosan + PDF 5 mg 5b 10b 
LPS1c + PDF 0.1 mg 10 
LPS2d + PDF 0.1 mg 10 
CVFe/zymosan + PDF 25 Ue/5 mg 10 
Vehicle/zymosan + PDF 0.5 ml/5 mg 10 
Crry-Igf/zymosan + PDF 1.2 mg/5 mg 10 
sCR1g/zymosan + PDF 3 mg/5 mg 10 
10 Zymosan + PDFf None/5 mg 10 
GroupTreatment (i.p.)Amount (mg/rat/each i.p.)Volume and Timing of i.p. Treatment (ml)
Day 0Day 1Days 2–4
PDFa only — 10 
Zymosan + PDF 5 mg 10 
Zymosan + PDF 5 mg 5b 10b 
LPS1c + PDF 0.1 mg 10 
LPS2d + PDF 0.1 mg 10 
CVFe/zymosan + PDF 25 Ue/5 mg 10 
Vehicle/zymosan + PDF 0.5 ml/5 mg 10 
Crry-Igf/zymosan + PDF 1.2 mg/5 mg 10 
sCR1g/zymosan + PDF 3 mg/5 mg 10 
10 Zymosan + PDFf None/5 mg 10 
a

4.25% acid-base PDF.

b

PDF was i.p. injected without zymosan.

c

LPS1; S. typhimurium.

d

LPS2; E. coli 055:B5.

e

Twenty-five units of CVF in 0.5 ml of sterile isotonic saline was i.v. injected on days −1 and 2.

f

Crry-Ig (or PDF only as the vehicle) was injected on days 0, 2, and 4.

g

sCR1 was injected on days 0, 1, 2, 3, and 4.

To investigate the relationship between tissue injury and complement activation in our zymosan-triggered peritonitis model, 25 U of cobra venom factor (CVF) in 0.5 ml of sterile isotonic saline was i.v. administered 24 h before surgery and again on day 2 in rats subjected to the group 2 protocol described above (group 6, n = 6; Table I). This schedule of CVF administration was planned from our previous data showing that systemic complement was depleted for at least 72 h following a single 25-U CVF injection (31). Systemic complement suppression was confirmed by measuring serum complement hemolytic activities as described previously (25). As the control, the same amount of sterile isotonic saline was injected in another group of rats subjected to the group 2 protocol (group7, n = 6; Table I).

Furthermore, to investigate the late peritoneal changes occurring in animals in which early-phase changes were influenced by complement depletion using CVF, we repeated the studies described above as groups 6 and 7 but sacrificed the new groups of rats and analyzed the peritoneum on days 18 and 36 (n = 6 for each group and each time course).

Crry-Ig was injected i.p. to investigate the effects of local inhibition of complement activation in the development of zymosan-induced severe peritoneal inflammation. Inhibition of complement activation was found for up to 53 h after a systemic single injection of Crry-Ig (20 mg/kg) as described in our previous report (30); in the current study, we i.p. injected 1.5 mg of Crry-Ig per animal in 0.5 ml of sterile PBS mixed with the PDF in the scraped peritonitis model just after the operation and every 2 days thereafter, Rats also received daily zymosan injections for 5 days after scraping (group 8 in Table I, n = 6). In another group subjected to the same scrape and zymosan treatment, soluble complement receptor 1 (sCR1; also called TP10, a gift from T Cell Sciences (now Avant Immunotherapeutics) was i.p. administered (3 mg/animal/day) instead of Crry-Ig (group 9 in Table I, n = 3). Control rats were i.p. injected with vehicle in PDF (group 10, n = 7). All rats in these protocols were killed on day 5 and parietal peritoneal tissue and blood were harvested as described above.

In a separate experiment using the same protocol as in group 2, we i.p. injected 0.1 mg of S. typhimurium (LPS 1; group 4) or 0.1 mg of E. coli (LPS2; group5) LPS as a surrogate for the respective microorganisms and instead of zymosan. On day 5, we harvested parietal peritoneum to analyze macroscopic, LM, and immunohistochemical effects. In this experiment, we included additional sets of control (PDF treated as in group 1) and zymosan-treated (as in group 2) rats. The number of rats in each group is displayed (see Fig. 10). A dose of 0.1 mg of LPS per animal was chosen because, as in our previous report (32), some rats that received 1 mg of LPS promptly died.

Harvested parietal peritoneum was fixed in 10% buffered formalin and embedded in paraffin. Sections (3-μm thick) were stained with H&E and Masson’s trichrome (MT) for histological analysis. Under LM, we scored sections. First, thickness of the subperitoneal compact zone, which was measured from the surface of the peritoneum to the edge of the subserosal structure, excluding the muscle layer, was randomly measured at five independent points of each section, and the mean was calculated and used as the value of peritoneal thickness for each rat. Second, we estimated fibrosis in parietal peritoneum using MT stain under LM.

Harvested parietal peritoneum was snap frozen in tissue compound, sectioned in a cryostat at 3 μm, and fixed in acetone at room temperature for 5 min. To observe the deposition of C3b and MAC, fixed sections were incubated with the optimal dilution (15 μg/ml) of pc anti-rat C3 (Cappel Labs) or in-house pc anti-rat C9 (22, 25, 26), followed by FITC-labeled goat anti-rabbit IgG Ab (1/200; Cappel Labs) absorbed with normal rat serum (1:1, v/v). We also examined C3b deposition using mAb C3/30, followed by FITC-labeled goat anti-mouse IgG (Cappel Labs) absorbed with normal rat serum (1:1, v/v). To observe fibrin exudation in peritoneum, sections were stained with FITC-labeled rabbit anti-human fibrinogen cross-reactive against rat fibrinogen (DakoCytomation).

To examine the distribution of the CReg Crry, CD55, and CD59, mAbs (TLD1C11, RDIII-7, and 6D1, respectively) were first incubated with the tissue sections, followed by FITC-labeled anti-mouse IgG.

Immunofluorescence staining intensities for each CReg, C3, MAC, C3b, and fibrinogen were graded into six categories: negative (−), very weak or trace (±), weak (+), moderate (++), strong (+++), and very strong (++++).

To investigate accumulated inflammatory cells, we counted total leukocytes and ED-1-positive cells. For these immunohistochemical analyses, we used a N-Histofine Simple stain MAX-PO (M) kit (Nichirei Bioscience) according to the manufacturer’s information. mAb ED-1 to detect rat monocytes/macrophage (BMA Biomedicals) was incubated on deparaffinized sections pretreated with 0.3% H2O2 to block endogenous peroxidase. mAb anti-leukocyte common Ag (LCA; clone OX1; Dainippon Pharmaceutical) to detect total leukocytes was incubated on fresh frozen sections pretreated with 0.3% H2O2 to block endogenous peroxidase, followed by HRP-labeled goat anti-mouse IgG. Development for ED-1- positive cells was performed using diaminobenzidine tetrahydrochloride development reagent (SAB-PO (M); Nichirei Bioscience) and development for LCA-positive cells was performed using a N-Histofine Simple Stain AEC Solution (Nichirei Bioscience). Finally, counterstaining was performed with hematoxylin.

The number of positive cells was counted in each frame under ×200 magnification. Twenty random frames were estimated and the mean was taken as the value of LCA-positive cells and ED-1 positive cells for each rat.

All values are expressed as the mean ± SEM. Statistical analysis was performed by one-factor ANOVA. When significant differences were present, statistical analysis was further performed using Scheffe’s F test between two groups. Significance between two groups was claimed when p < 0.05 (5%).

Before the following experiments, we first compared peritoneal injury generated by daily administration of 0.1, 1, and 5 mg of zymosan per animal for up to 5 days in the scraped rat model. All rats survived, even at the highest dose. On day 5 after administration of each zymosan dose, obvious macroscopic changes were observed across the whole peritoneum in rats receiving five-daily 5-mg doses and mild and segmental macroscopic changes in rats receiving 0.1 or 1 mg of zymosan per animal per day. The peritoneal thickness and number of inflammatory cells were dependent on the dose of zymosan (data not shown). Based on these observations, a dose of 5 mg of zymosan per animal was chosen for subsequent studies. All rats treated with 2 mg of LPS per animal died, as reported previously (32); a dose of 1 mg of LPS per animal caused death in a variable proportion of the rats; and at a dose of 0.1 mg/animal, all rats survived. Therefore, we chose a dose of 0.1 mg of LPS per animal for subsequent experiments.

Macroscopic findings in group 2 rats at day 3 comprised a few small white plaques (black arrows in Fig. 1,C); no plaques were found in group 1 rats (Fig. 1,A). At that time, the parietal peritoneum in both groups presented a smooth surface. By day 5, a smooth, white fibrous membrane was present on the surface of the scraped parietal peritoneum (group 1; Fig. 1,E). In contrast, on day 5 in rats given five daily i.p. doses of zymosan (group 2), there were abundant yellow-white plaques on the surface of the parietal peritoneum; some fused to form large plaques surrounded by a reddish swollen zone in the parietal peritoneum (black arrows in Fig. 1,G). On day 18, the peritoneum in group 1 rats appeared grossly normal, while in group 2 rats, plaque fusion had continued to create a yellow-white fused sheet covering the peritoneum (black arrows in Fig. 1,K), with numerous small vessels running into the plaques (black arrowheads in Fig. 1,K), suggesting peritoneal neovascularization. In group 2 rats on day 36, subperitoneal vessels were obscured, suggesting peritoneal thickening (white arrowheads in Fig. 1,O). In group 1 rats from day 18 onward, the parietal peritoneum was of normal appearance (Fig. 1, I and M).

FIGURE 1.

Macroscopic and microscopic findings with or without zymosan injections in peritoneal injury model. Frames A, B, E, F, I, J, M, and N are from group 1 rats (control) and frames C, D, G, H, K, L, O, and P are from group 2 (zymosan). Left frame of each set shows the macroscopic appearance of parietal peritoneum. Right side of each set shows the microscopic appearance stained with H & E. The original magnifications were ×100. The timing of sacrifice is displayed on the left side for each set. Arrows in frame C indicate small plaques. On day 5, fusion of the plaques was observed, arrowed around red swelling (magnified and shown with arrow in right bottom corner). On day 18, the surface of peritoneum was covered with white fibrous tissue and subperitoneal vessels were obscured (arrowheads in frame K). Fibrous tissue formation was accompanied with neoangiogenesis (arrowheads in right bottom corner in frame K). White arrowhead shows vessels obscured under thickened peritoneum on day 36 (frame O). ∗, External face of peritoneum. Scale bars are in the upper left corner of frame A for macroscopic appearance and B for microscopic appearance.

FIGURE 1.

Macroscopic and microscopic findings with or without zymosan injections in peritoneal injury model. Frames A, B, E, F, I, J, M, and N are from group 1 rats (control) and frames C, D, G, H, K, L, O, and P are from group 2 (zymosan). Left frame of each set shows the macroscopic appearance of parietal peritoneum. Right side of each set shows the microscopic appearance stained with H & E. The original magnifications were ×100. The timing of sacrifice is displayed on the left side for each set. Arrows in frame C indicate small plaques. On day 5, fusion of the plaques was observed, arrowed around red swelling (magnified and shown with arrow in right bottom corner). On day 18, the surface of peritoneum was covered with white fibrous tissue and subperitoneal vessels were obscured (arrowheads in frame K). Fibrous tissue formation was accompanied with neoangiogenesis (arrowheads in right bottom corner in frame K). White arrowhead shows vessels obscured under thickened peritoneum on day 36 (frame O). ∗, External face of peritoneum. Scale bars are in the upper left corner of frame A for macroscopic appearance and B for microscopic appearance.

Close modal

We also compared rats given five doses of zymosan over 5 days (group 2) with rats given just one dose of zymosan on day 0 (group 3). A single zymosan injection did not induce significant plaque formation at day 5 or later times and the gross appearance resembled that in group 1 rats.

Pathological changes in both groups 1 and 2 were assessed across the time course using LM; a thin inflammatory cell layer was found in both groups 1 and 2 on day 1 (data not shown). On day 3, the inflammatory cell layer was increased compared with day 1, accompanied by edematous changes under the peritoneal surface in both groups 1 and 2 (Fig. 1, B and D). At that time, the changes were not significantly different between the groups (Fig. 2,A). In group 2 rats on days 5 and 18, there was progressive accumulation of inflammatory cells and increased thickness of the parietal peritoneum (Figs. 1, H and L, and 2). On day 18, small vessels were observed under the peritoneum (arrowheads in the right bottom corner of Fig. 1,K). On day 36, peritoneal thickening and accumulation of inflammatory cells in the subperitoneal layer were still observed in group 2 rats (Figs. 1,P and 2,A). In contrast, from day 5 onward in group 1 rats, subperitoneal accumulation of inflammatory cells was markedly decreased compared with day 1 and there was no significant peritoneal thickening (Figs. 1, F, J, and N, and 2 A).

FIGURE 2.

Subperitoneal thickness and LCA- and ED-1-positive cells in the peritoneum with or without zymosan injections in the peritoneal injury model. The graphs show the pooled data from zymosan-treated (group 2; zymosan) and control (group 1; control) rats. Thickness of peritoneum (A), LCA-positive cell infiltration (B), and ED-1 positive cell infiltration (C) in the peritoneum were all significantly increased in group 2 compared with group 1.

FIGURE 2.

Subperitoneal thickness and LCA- and ED-1-positive cells in the peritoneum with or without zymosan injections in the peritoneal injury model. The graphs show the pooled data from zymosan-treated (group 2; zymosan) and control (group 1; control) rats. Thickness of peritoneum (A), LCA-positive cell infiltration (B), and ED-1 positive cell infiltration (C) in the peritoneum were all significantly increased in group 2 compared with group 1.

Close modal

We compared the effects of a single zymosan injection (group 3) with five sequential daily injections (group 2) on histological appearance. Accumulation of inflammatory cells and thickening of subserosal tissues was observed in group 2 rats, replicating the results in experiment 1; in contrast, group 3 rats showed no significant enhancement of inflammation or peritoneal thickening and resembled group 1 in experiment 1 (Fig. 3). Those findings support the macroscopic observations in these groups described above.

FIGURE 3.

Comparison of repeated (group 2) and single (group 3) zymosan injections in the peritoneal injury model. Frames A and B show representative sections of the peritoneum stained by H & E. ∗, External face of the peritoneum. The original magnifications were ×100. Scale bar is in the upper left corner of frame A. Frames C–E show the averaged data for each group; thickness of subperitoneum, infiltrating LCA-positive cells, and infiltrating ED-1 positive cells, respectively. Significance of differences are shown for each data set.

FIGURE 3.

Comparison of repeated (group 2) and single (group 3) zymosan injections in the peritoneal injury model. Frames A and B show representative sections of the peritoneum stained by H & E. ∗, External face of the peritoneum. The original magnifications were ×100. Scale bar is in the upper left corner of frame A. Frames C–E show the averaged data for each group; thickness of subperitoneum, infiltrating LCA-positive cells, and infiltrating ED-1 positive cells, respectively. Significance of differences are shown for each data set.

Close modal

We quantified the severity of pathology of scraped parietal peritoneum by measuring the subperitoneal thickness in group 1 and 2 rats. The subperitoneal thickness on days 1 and 3 was similar between groups 1 and 2. However, from day 5 onward the peritoneal thickness in group 2 was clearly increased compared with that of group 1 and progressively increased despite cessation of zymosan treatment on day 4 (Fig. 2 A).

Peritoneal fibrosis was minimal on days 3 and 5 in both groups 1 and 2 (Figs. 4, A–D). In group 1 rats on days 18 and 36, most of the subperitoneal compact zone was occupied with fibrous tissues (Fig. 4, E and G), although the subperitoneal thickness was decreased after day 5 (Fig. 2,A), suggesting that the subserosa was replaced with fibrous tissue. In contrast, in group 2 rats, cellular components were predominant in proliferative peritoneal tissues on days 18 and 36 (Fig. 4, F and H). Comparison between groups 1 and 2 of inflammatory cell infiltration in the peritoneum on days 1 and 3 showed no significant differences, the majority of inflammatory cells were LCA positive and ED-1 negative (Fig. 2, B and C). In group 2 rats on day 5, both LCA-positive and ED-1-positive cells were increased and remained high on days 18 and 36 (Fig. 2, B and C). In contrast, the numbers of LCA-positive and ED-1-positive cells in group 1 peaked on day 3 and after that decreased and were significantly less than those of group 2 (Fig. 2, B and C).

FIGURE 4.

Fibrosis and cellular proliferations in peritoneum with and without zymosan in the peritoneal injury model. Frames A, C, E, and G show representative peritoneal pathology in group 1 rats (control) and frames B, D, F, and H show representative pathology in group 2 rats (zymosan). The sections were subjected to MT staining. Blue shows fibrous tissues and red shows cellular proliferating tissues. Original magnifications were ×100. Scale bar is in the upper left corner of frame A. ∗, External face of the peritoneum.

FIGURE 4.

Fibrosis and cellular proliferations in peritoneum with and without zymosan in the peritoneal injury model. Frames A, C, E, and G show representative peritoneal pathology in group 1 rats (control) and frames B, D, F, and H show representative pathology in group 2 rats (zymosan). The sections were subjected to MT staining. Blue shows fibrous tissues and red shows cellular proliferating tissues. Original magnifications were ×100. Scale bar is in the upper left corner of frame A. ∗, External face of the peritoneum.

Close modal

When the number of peritoneal inflammatory cells was compared between rats given five daily zymosan doses (group 2) or a single zymosan dose (group 3) on day 0, the number of inflammatory cells in group 2 was clearly higher than that in group 3 on day 5 (n = 6 for each group, p < 0.0001 in counts of LCA-positive cells and p < 0.05 in counts of ED-1-positive cells; Fig. 3, D and E).

In normal rat peritoneum, Crry, CD55, and CD59 were all strongly expressed on the mesothelial layer of the peritoneum, whereas expression in the subperitoneal compact zone was weak (Fig. 5, A, C, and D). On day 1 after peritoneal scrape in group 1 and 2 rats, expression of Crry, CD55 and CD59 was clearly decreased on the parietal peritoneum in the scraped area, although expression in the subperitoneal area, such as on small vessels, was preserved (Table II). CReg expression was further decreased in group 2 on day 5 (Fig. 5, M, O, and P), although the CReg expression in the mesothelial layer was recovering in group 1 rats on day 5 (Fig. 5, E, G, and H). Expression of Crry, CD55, and CD59 in the mesothelial layer of the peritoneal surface had been recovering in group 2 rats on days 18 and 36 (Fig. 5, Q, S, and T). In group 2, many CReg-positive inflammatory cells were observed in the submesothelial compact zone.

FIGURE 5.

Expression of membrane complement regulators (CRegs) in the peritoneum in untreated rats, groups 1 and 2 rats on days 5 and 36. Sections were stained for Crry (A, E, I, M, and Q), CD46 (B, F, J, N, and R), CD55 (C, G, K, O, and S), or CD59 (D, H, L, P, and T). A–D show sections from untreated rat peritoneum. E–L and M–T are peritoneum of groups 1 (control) and 2 rats (zymosan), respectively. E–H and M–P show peritoneum on day 5 and I–L and Q–T on day 36. Exposure times and other staining and imaging parameters were kept constant to permit comparison. ∗, External face of the peritoneum. Original magnification was ×400 for frames A–D and ×200 for frames E–T. Scale bars are in the upper left corner of frames A and E.

FIGURE 5.

Expression of membrane complement regulators (CRegs) in the peritoneum in untreated rats, groups 1 and 2 rats on days 5 and 36. Sections were stained for Crry (A, E, I, M, and Q), CD46 (B, F, J, N, and R), CD55 (C, G, K, O, and S), or CD59 (D, H, L, P, and T). A–D show sections from untreated rat peritoneum. E–L and M–T are peritoneum of groups 1 (control) and 2 rats (zymosan), respectively. E–H and M–P show peritoneum on day 5 and I–L and Q–T on day 36. Exposure times and other staining and imaging parameters were kept constant to permit comparison. ∗, External face of the peritoneum. Original magnification was ×400 for frames A–D and ×200 for frames E–T. Scale bars are in the upper left corner of frames A and E.

Close modal
Table II.

Deposition of complement activation products and distributions of CRegs in group 1 and 2 rats

GroupDayC3/C3bMACCrryCD55CD59Fibrinogen
++/++ ± − − − ++ 
+/+ ± ± ± ± ++ 
±/± − ± 
18 −/− − − 
36 −/− − − 
+++/+++ − − − ++ 
+++/+++ ± ± ± ++ 
++/++ ++ ++ +++ 
18 +/+ ± ++ ++ 
36 +−/±/± ± ++ ++ ± 
GroupDayC3/C3bMACCrryCD55CD59Fibrinogen
++/++ ± − − − ++ 
+/+ ± ± ± ± ++ 
±/± − ± 
18 −/− − − 
36 −/− − − 
+++/+++ − − − ++ 
+++/+++ ± ± ± ++ 
++/++ ++ ++ +++ 
18 +/+ ± ++ ++ 
36 +−/±/± ± ++ ++ ± 

Deposition of C3b was observed on the injured peritoneal surface in both groups 1 and 2 (arrows in Figs. 6, A-1, A-2, B-1, and B-2). On day 1, MAC deposition was abundant in group 2 rats (arrows in Fig. 6,B-3) but trace in group 1 rats (Fig. 6,A-3). On day 5, C3b and MAC deposition was clearly found in the peritoneum in group 2 rats (Fig. 6, F-1–F-3). In contrast, those complement activation products were absent or trace in group 1 rats on day 5 (Fig. 6, E-1–E-3). On days18 and 36, small amounts of C3b and MAC remained in subperitoneal tissues in group 2 (Fig. 6, H-1–H-3 and J-1–J-3) but not in group 1 (Fig. 6, G-1–G-3 and I-1–I-3).

FIGURE 6.

Deposition of activated complement components, C3b and MAC, in the peritoneum with and without zymosan in the peritoneal injury model. Sections were stained for C3 (each photoset suffix “-1”), C3b (each photoset suffix “-2”), and MAC (each photoset suffix “-3”). Photosets A, C, E, G, and I are from group 1 rats (control) and B, D, F, H, and J from group 2 rats (zymosan). Photosets A and B are from day 1, C and D from day 3, E and F from day 5, G and H from day 18, and I and J from day 36. Arrows indicate C3 or MAC deposition. Exposure times and other parameters were kept constant to permit comparison. ∗, External face of the peritoneum. Original magnification was ×200. Scale bar is in the upper left corner of frame A-1.

FIGURE 6.

Deposition of activated complement components, C3b and MAC, in the peritoneum with and without zymosan in the peritoneal injury model. Sections were stained for C3 (each photoset suffix “-1”), C3b (each photoset suffix “-2”), and MAC (each photoset suffix “-3”). Photosets A, C, E, G, and I are from group 1 rats (control) and B, D, F, H, and J from group 2 rats (zymosan). Photosets A and B are from day 1, C and D from day 3, E and F from day 5, G and H from day 18, and I and J from day 36. Arrows indicate C3 or MAC deposition. Exposure times and other parameters were kept constant to permit comparison. ∗, External face of the peritoneum. Original magnification was ×200. Scale bar is in the upper left corner of frame A-1.

Close modal

The detailed staining profiles of CRegs and complement deposition were semiquantified and are shown in Table II.

Fibrinogen was strongly detected on the injured peritoneal surface in both groups 1 and 2 on days 1 and 3 (Fig. 7, A–D). In group 2 rats treated with zymosan, strong fibrinogen deposition was still observed on the peritoneum at days 5 and 18 (Fig. 7, F and H) and small deposits of fibrinogen still remained along the peritoneal surface and subperitoneal layer on day 36 (Fig. 7,J). In contrast, in group 1 rats without zymosan treatment, fibrinogen was not detected after day 5 (Fig. 7, E, G, and I). Those findings suggested that fibrinogen exudation as an aspect of acute inflammation was enhanced and progressed by zymosan stimulation.

FIGURE 7.

Exudation of fibrinogen in the peritoneum with and without zymosan treatment in the peritoneal injury model. A, C, E, G, and I show sections from group 1 rats without zymosan treatment (control) and B, D, F, H, and J from group 2 rats with zymosan treatment (zymosan). On days 1 and 3, both groups 1 and 2 rats showed strong fibrinogen deposition in the peritoneum (arrows). In group 2 on days 5, 18, and 36, fibrinogen was strongly deposited in the peritoneum (arrows), although deposition of fibrinogen was minimal or absent in group 1 on those days. ∗, External face of the peritoneum. Original magnification was ×200. Scale bar is in the upper left corner of frame A.

FIGURE 7.

Exudation of fibrinogen in the peritoneum with and without zymosan treatment in the peritoneal injury model. A, C, E, G, and I show sections from group 1 rats without zymosan treatment (control) and B, D, F, H, and J from group 2 rats with zymosan treatment (zymosan). On days 1 and 3, both groups 1 and 2 rats showed strong fibrinogen deposition in the peritoneum (arrows). In group 2 on days 5, 18, and 36, fibrinogen was strongly deposited in the peritoneum (arrows), although deposition of fibrinogen was minimal or absent in group 1 on those days. ∗, External face of the peritoneum. Original magnification was ×200. Scale bar is in the upper left corner of frame A.

Close modal

Complement was systemically depleted by CVF administration before initiating the model (group 6); controls were injected with vehicle (group 7). In group 6 on day 5, the surface of the parietal peritoneum was macroscopically smooth with no plaques in four of seven rats; three of seven in this group had small plaques on day 5. In contrast, group 7 rats all had large peritoneal plaques on day 5 (Fig. 8,D). Pathologically, the peritoneal thickening on day 5 in group 6 rats was significantly less than in that in group 7 rats (Fig. 8,G). Moreover, the number of inflammatory cells in group 6 was significantly suppressed compared with that of group 7 (Fig. 8, H and I). Of note, the plaque-free rats in group 6 showed minimal staining for C3b and MAC (Fig. 8, B and C), whereas the plaque-positive rats in group 6 had segmental C3b and MAC deposition around the peritoneal surface (data not shown).

FIGURE 8.

Systemic complement depletion by CVF suppresses peritoneal inflammation. A–C show sections from group 6 rats (CVF treated; CVF) and D–F show sections from group 7 rats (vehicle; control) on day 5. A and D are H & E-stained micrographs on day 5. B and E show C3 deposition. C and F show MAC deposition on day 5. ∗, External face of the peritoneum. Original magnification was ×100 for frames A and D and ×200 for frames B, C, E, and F. Scale bars are in the upper left corner of frames A and B. G–I show the average data for each group measuring thickness of peritoneum, infiltrating LCA-positive cells, and infiltrating ED-1-positive cells, respectively, in groups 6 and 7 rats on days 5, 18, and 36. Significance of differences between groups are shown in each frame.

FIGURE 8.

Systemic complement depletion by CVF suppresses peritoneal inflammation. A–C show sections from group 6 rats (CVF treated; CVF) and D–F show sections from group 7 rats (vehicle; control) on day 5. A and D are H & E-stained micrographs on day 5. B and E show C3 deposition. C and F show MAC deposition on day 5. ∗, External face of the peritoneum. Original magnification was ×100 for frames A and D and ×200 for frames B, C, E, and F. Scale bars are in the upper left corner of frames A and B. G–I show the average data for each group measuring thickness of peritoneum, infiltrating LCA-positive cells, and infiltrating ED-1-positive cells, respectively, in groups 6 and 7 rats on days 5, 18, and 36. Significance of differences between groups are shown in each frame.

Close modal

To investigate effects of local suppression of complement activation in the peritoneum, we tested two inhibitors, Crry-Ig and sCR1. Local suppression of complement activation in the peritoneum was achieved by i.p. injection of Crry-Ig every 2 days (group 8) or by daily i.p. injection of sCR1 (group 9), each started before initiating the model. Local i.p. administration of Crry-Ig and sCR1 at these doses causes insignificant systemic complement inhibition (data not shown). In these groups, the surface of the parietal peritoneum remained smooth compared with that of controls (group 10) injected with vehicle (data not shown). Under LM, subperitoneal thickening was much reduced compared with that of controls (Fig. 9, A, D, and G). In group 8, C3b and MAC were not found along the peritoneum in any of the rats (Fig. 9, B and C). In group 9, trace C3b deposition was found along the peritoneum (Fig. 9,E) but deposition of MAC was minimal (Fig. 9,F). In contrast, controls in group 10 had strong peritoneal C3b and MAC deposition as seen in group 2 rats (Fig. 9, H and I). Deposition of fibrinogen was also suppressed by systemic CVF treatment, local Crry-Ig treatment, and local sCR1 (data not shown). The results confirm that the zymosan enhancement of peritoneal injury following mechanical scraping was critically dependent on peritoneal complement activation.

FIGURE 9.

Intraperitoneal complement inhibition by either Crry-Ig or sCR1 prevents peritoneal proliferation. Frames show representative peritoneal sections from group 8 rats (Crry-Ig; A–C), group 9 rats (sCR1; D–F), and group 10 rats (control; G–I) stained with H & E (A, D, and G) for C3 deposition (B, E, and H) or MAC deposition (C, F, and I). Original magnification was ×200 for frames A, D, and G and ×100 for frames B, C, E, F, H, and I. ∗, External face of the peritoneum. Scale bars are in the upper left corner of frames A and B. Pooled data from the groups in J–L show thickness of peritoneum, infiltrating LCA-positive cells, and infiltrating ED-1-positive cells, respectively, in groups 8–10 rats. Significance of differences between the groups are shown in J–L.

FIGURE 9.

Intraperitoneal complement inhibition by either Crry-Ig or sCR1 prevents peritoneal proliferation. Frames show representative peritoneal sections from group 8 rats (Crry-Ig; A–C), group 9 rats (sCR1; D–F), and group 10 rats (control; G–I) stained with H & E (A, D, and G) for C3 deposition (B, E, and H) or MAC deposition (C, F, and I). Original magnification was ×200 for frames A, D, and G and ×100 for frames B, C, E, F, H, and I. ∗, External face of the peritoneum. Scale bars are in the upper left corner of frames A and B. Pooled data from the groups in J–L show thickness of peritoneum, infiltrating LCA-positive cells, and infiltrating ED-1-positive cells, respectively, in groups 8–10 rats. Significance of differences between the groups are shown in J–L.

Close modal

To observe the later peritoneal injuries occurring after systemic complement depletion with CVF treatment (group 6), the peritoneal thickness and the number of inflammatory cells were also observed in separate experimental groups on days 18 and 36, i.e., after complement levels have recovered posttreatment (Fig. 8, G–I). Compared with CVF-treated rats on day 5, the peritoneal thickness and inflammatory cell number was increased on day 18, but decreased by day 36, reflecting delayed injury and recovery. At both days 18 and 36 in rats without CVF treatment (group 7), inflammation as assessed by peritoneal thickness and number of inflammatory cells followed the same trend but was clearly worse than in CVF-treated rats.

To investigate whether induction of severe peritoneal inflammation was unique to zymosan, we tested the effects of the complement-activating bacterial product LPS. Two different types of LPS (groups 4 and 5) were administered and compared with zymosan administration in the peritoneal scrape model (group 2). Macroscopic examination revealed no plaques at day 5 in groups 4 and 5 rats, compared with marked plaque formation in group 2 rats (data not shown). LM analysis of groups 4 and 5 rats on day 5 showed mild thickening of the submesothelial compact zone and accumulation of inflammatory cells of LCA-positive cells and of ED1-positive cells to a similar degree to that in the control group 1 rats and markedly less than in group 2 (Fig. 10, E–G). Deposition of C3b and MAC in groups 4 and 5 rats was limited to the peritoneal surface (arrows in Fig. 10, B-2 and C-2) and was less than the deposition in group 2 rats (Fig. 10 A-2).

FIGURE 10.

Comparison of effects of zymosan and LPS in the peritoneal injury model. A–D show representative peritoneal sections from rats treated with zymosan (group 2; zymosan), LPS of S. typhimurium (group 4; LPS1), LPS of E. coli (group 5; LPS2), or vehicle (group 1; control) in the model, respectively. Sections were stained for H & E (each photoset suffix “-1”), C3b (each photoset suffix “-2”), and MAC (each photoset suffix “-3”). ∗, External face of the peritoneum. Original magnification was ×100 for H & E staining and ×200 for C3b and MAC staining. Scale bars are in the upper left corner of frames A-1 and A-2. Pooled data from the groups are shown in E–G for thickness of peritoneum, infiltrating LCA-positive cells, and infiltrating ED-1 positive cells, respectively. Significance of differences between the groups are shown.

FIGURE 10.

Comparison of effects of zymosan and LPS in the peritoneal injury model. A–D show representative peritoneal sections from rats treated with zymosan (group 2; zymosan), LPS of S. typhimurium (group 4; LPS1), LPS of E. coli (group 5; LPS2), or vehicle (group 1; control) in the model, respectively. Sections were stained for H & E (each photoset suffix “-1”), C3b (each photoset suffix “-2”), and MAC (each photoset suffix “-3”). ∗, External face of the peritoneum. Original magnification was ×100 for H & E staining and ×200 for C3b and MAC staining. Scale bars are in the upper left corner of frames A-1 and A-2. Pooled data from the groups are shown in E–G for thickness of peritoneum, infiltrating LCA-positive cells, and infiltrating ED-1 positive cells, respectively. Significance of differences between the groups are shown.

Close modal

Pathological changes in the peritoneum, such as peritoneal thickening with expansion of the submesothelial compact zone, increase of small vessels, and fibrosis, have been reported after long-term exposure to PDF (14). As a consequence, insufficiency of ultrafiltration and dialysis is common after long-term PD therapy. Another problem causing pathological changes in the peritoneum is infectious peritonitis, especially when recurrent, which can also homeostasis damage in the peritoneum, enhance peritoneal injury and accelerate peritoneal sclerosis. Both long-term PD therapy and infection play roles in the development of EPS in the PD patient. Fungal infections cause severe infectious peritonitis related to PD therapy, although they are not common (33). Peritoneal fungal infection provokes consideration of withdrawal of PD therapy in the patient according to recent guidelines (5), because of the poor prognosis and risk of development of EPS. Indeed, the incidence of EPS increased when PD therapy was continued for >5 years (34), when recurrent infectious peritonitis occurred (35) or when fungal infection occurred (35). Fibrosis and/or angiogenesis in EPS was associated with IL-8 and TGF-β and these were reported to be important mediators for development of EPS in PD patients (36, 37). However, little is known about the triggers for chronic peritoneal damage, including fungal infections. Moreover, it remains unclear why EPS can still progress even after stopping PD and after removing the PD catheter in patients with postfungal peritonitis.

We recently described a mechanical peritoneal injury model that temporarily increased TGF-β production and induced peritoneal fibrosis in rats within 14 days (19). In this study, we show that zymosan, which is a cell wall component of fungus, caused proliferative peritonitis with severe accumulation of inflammatory cells when given in conjunction with the mechanical injury. Repeated administration of zymosan was necessary to cause severe inflammation which was thereafter sustained even weeks after cessation of zymosan treatment. In contrast, repeated administration of two different types of LPS did not enhance peritonitis in the model, providing a possible explanation for the observed poor prognosis of fungal peritonitis in PD patients. Deposition of fibrinogen, which is a marker of severe and acute inflammation in tissues, was clearly enhanced in zymosan-treated rats and continued even on day 36.

Because zymosan is a powerful activator of complement, we examined roles of complement activation in the development of the zymosan-related peritonitis. In the initial phase of the model, decreased expression of CRegs was observed with deposition of complement activation products. On day 5, loss of CReg expression was still apparent in zymosan-treated rats, but recovered without zymosan in the model. Complement activation products C3b and MAC were present on days 1 and 3 in zymosan-treated and untreated rats, but after day 5, complement deposition was found only in the zymosan-treated animals, persisting through to day 36.

Severe peritonitis in zymosan-treated rats was significantly suppressed by systemic complement depletion with CVF, confirming the role of complement. Intraperitoneal administration of two different complement inhibitors, Crry-Ig fusion protein and sCR1, also suppressed peritoneal inflammation and thickening. Crry-Ig has a longer half-life in vivo compared with sCR1, reducing the number of i.p. administrations required (30). Both therapies were effective, local Crry-Ig administration rather better than local sCR1 administration, likely because of better pharmacokinetics. Peritoneal deposition of C3b and MAC was clearly suppressed by either systemic or local inhibition of complement.

The data suggest that the severe peritoneal injury induced by fungal infection is caused by complement activation and anticomplement therapy might be useful to prevent the peritoneal damage. The model also showed that acute peritoneal fungal infection, modeled by 5 days of zymosan administration, could develop into progressive and proliferative inflammation and induce chronic peritoneal damage. The initial complement activation was essential to develop these later peritoneal changes.

Of note, late peritoneal inflammation, sampled at days 18 and 36, was present even after the initial inflammation had been suppressed by CVF treatment, reflecting the fact that complement depletion delayed but did not ablate the injury. Activation of complement at these late times might suggest that zymosan remained present in the peritoneum; however, the severity of peritoneal inflammation on days 18 and 36 was clearly less in rats that had received initial complement suppression, suggesting that there had been at least partial clearance of the zymosan trigger.

To test whether other complement-activating pathogen products similarly enhanced damage in the model, we compared zymosan with two different types of LPS. Although zymosan induced severe inflammatory peritoneal proliferation, LPS from either S. typhimurium or E. coli caused no significant enhancement of injury. One reason might be that zymosan is a better complement activator, because deposition of complement activation products in the peritoneum of zymosan-treated rats was much greater than in LPS-treated animals. However, zymosan is not only a strong complement activator but also induces inflammatory responses dependent on TLR 2 which are enhanced by engagement of the dectin-1 receptor (38). LPS also triggers complement activation and engages TLRs, predominantly but mainly related to TLR4 (39), although bacterial lipoproteins can engage TLR2 (40). Pulmonary inflammation triggered by installation of zymosan was recently shown to be independent of complement, TLRs and dectin-1, indicating that zymosan also engages other inflammatory pathways (41). Zymosan might therefore enhance inflammation through mechanisms not shared with LPS; however, in the current model, complement activation is clearly the key difference.

Currently, there are no effective therapies to prevent and/or treat EPS, although immune-suppressive therapies such as tamoxifen and steroids were reported to improve EPS in humans (42, 43, 44). Spironolactone, angiotensin-converting enzyme inhibitor, and angiotensin II receptor blocker have also been reported to improve peritoneal fibrosis in animal models (19, 45). Our results provoke us to suggest that a radically different approach using anticomplement therapy might be useful in prevention or treatment of EPS.

We greatly appreciate H. Nishimura, Y. Suzuki, N. Suzuki, N. Asano, and Y. Fujitani for technical help.

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 work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry Education, Science, and Culture, Japan (no. 19590946) and the 2006 research grant from the Aichi Kidney Foundation. B.P.M. was supported by a Programme Grant from the Wellcome Trust (Grant 068590) and C.L.H. and N.J.H. were supported by the Wellcome Trust (Grant 068823) and the Welsh Office of Research and Development (Grant DTA01/1/014).

3

Abbreviations used in this paper: PD, peritoneal dialysis; EPS, encapsulating peritoneal sclerosis; PDF, PD fluid; CReg, complement regulator; MAC, membrane attack complex; LM, light microscope/microscopic/microscopy; CVF, cobra venom factor; sCR1, soluble complement receptor 1; MT, Masson’s trichrome; pc, polyclonal; LCA, leukocyte common Ag.

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