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181 Bull Vet Inst Pulawy 54, 181-187, 2010 EFFECT OF BREWERS’ YEAST (SACCHAROMYCES CEREVISIAE) EXTRACT ON SELECTED PARAMETERS OF HUMORAL AND CELLULAR IMMUNITY IN LAMBS ROMAN WÓJCIK Department of Microbiology and Clinical Immunology, Faculty of Veterinary Medicine, University of Warmia and Mazury in Olsztyn, 10-957 Olsztyn, Poland [email protected] Received for publication September 11, 2009 Abstract The objective of this study was to determine the stimulating effect of the brewers’ yeast Saccharomyces cerevisiae dietary supplement on selected parameters of specific and non-specific humoral and cellular immunity in lambs. The study involved 32 lambs aged 30 ±3 d, divided into two equal groups: control and experimental. Animals in the experimental group were fed a C-J concentrate mixed with a prebiotic, the extract of dried brewers’ yeast containing 10%-15% MOS and 25%-30% β-1,3/1,6-D-glucan in the amount of 3 g/kg of the concentrate. At the beginning of the experiment (day 0) and on the 15th, 30th, and 60th d of the study, blood was sampled from the jugular vein to determine γ-globulin levels, lysozyme and ceruloplasmin activity, proliferative response of blood lymphocytes (MTT) after stimulation with LPS or ConA, metabolic activity (RBA), and potential killing activity (PKA) of phagocytes. As regards humoral immunity parameters, significantly higher γ-globulin levels and higher lysozyme and ceruloplasmin activity were noted in the blood serum of experimental lambs supplemented with the yeast extract, in comparison with control lambs not fed the supplement. No statistically significant differences in serum total protein were found between the control and experimental groups. The analysis of cellular immunity indicators revealed significantly higher levels of RBA and PKA, and higher MTT rates after stimulation with LPS or ConA in the experimental group, in comparison with the control group. Key words: lambs, prebiotics, Saccharomyces cerevisiae, protein content, humoral immunity, cellular immunity. Owing to its prebiotic properties, Saccharomyces cerevisiae can be widely used as a natural productivity stimulator added to animals’ feed (9). The beneficial effects of yeast in the nutrition of ruminants have been noted in experiments on dairy cows (8) and suckling lambs (22). The above experiments also confirmed a stimulating effect of yeast on the health status of animals. The results of studies involving lambs (22, 35) indicate that β-1,3/1,6-D-glucan, a structural component of the cellular wall of Saccharomyces cerevisiae, plays an important role in this process, in regard to the indicators of both non-specific humoral immunity (19) and cellular immunity (35). The immunomodulating effect of Saccharomyces cerevisiae yeast is ascribed to mannan-oligosaccharides (MOS), which build the yeast's cell wall structure (19). By binding selected pathogens, MOS prevent them from colonising the host's gastrointestinal system and support their elimination by specialised immune cells (7, 12, 16). So far no studies have been conducted concerning the effect of dried yeast and yeast-derivative prebiotics on immunity indicators in lambs. The objective of this study was to determine the effect of the extract of dried brewers’ yeast Saccharomyces cerevisiae (Biolex-MB40) with an increased β-1,3/1,6-D-glucan and MOS content on selected parameters of humoral and cellular immunity in lambs. Material and Methods Experimental design. Thirty-two suckling Kamieniec breed lambs from a conservative herd, aged 30 ±3 d, were divided into two equal groups: I – control and II – experimental. Both groups were identical in terms of body weight on the second day of life, gender, birth type, as well as the age of the ewes, to eliminate possible differences in milk yield. Uniform feeding standards were applied in both groups in line with lamb nutrient requirements. Suckling lambs were fed haysilage of grass and legumes, and C-J concentrate. The quantity of administered feed and leftovers was monitored throughout the experiment. Experimental lambs were fed the Biolex–MB40 (Leiber GmbH) brewer's yeast (Saccharomyces cerevisiae) extract containing 10%-15% MOS and 25%-30% β-1,3/1,6-Dglucan. The extract was mixed with C-J concentrate in 182 the amount of 3 g/kg of the concentrate. C-J concentrate doses, identical for both groups, were increased every 10 d by 0.05 kg/animal/d, starting from 0.15 kg/animal/d. At the beginning of the experiment (day 0) and on the 15th, 30th, and 60th d of the study, blood was sampled from the jugular vein to determine selected parameters of humoral and cellular immunity. Evaluation of non-specific humoral immunity parameters. Lysozyme activity was determined by the turbidimetric method (25) modified by Siwicki and Anderson (29), ceruloplasmin activity – by the method developed by Siwicki and Studnicka (31), total protein content was determined by spectrophotometry as described by Lowry et al. (17) and modified by Siwicki and Anderson (29), and γ-globulin level was determined by the precipitation method modified by Siwicki and Anderson (29). Lysozyme activity. Whole blood samples were centrifuged for 5 min at 1,000 g to separate blood cells from the serum. The serum was diluted 1:1 with phosphate buffer, and 0.1 ml of the solution was placed in microplate wells. Next, 0.5 ml of Micrococcus lysodeikticus suspension (25 mg bacteria/100 ml of phosphate buffer) (Sigma Chemical Co.) was added. Absorbance was measured directly after the addition of bacteria (E0) and after 1, 2, 3, and 30 min (final E). The final absorbance was subtracted from the initial absorbance (E0) to determine lysozyme activity with the use of a standard curve. The standard curve was plotted based on the optical density values for known lysozyme concentrations. Ceruloplasmin activity. Whole blood samples were centrifuged for 5 min at 1,000 g to separate blood cells from the serum. The following buffers were prepared: 1) acetate buffer (pH 5.2, containing crystalline acetic acid, sodium acetate trihydrate, and 15 mg of EDTA), 2) buffered substrate solution (0.2% pphenyldiamine (PPD) in acetic buffer), 3) sodium azide solution (0.02% sodium azide solution in deionised water). 0.5 ml of buffered solution was added to each of two 16 x 100 mm test tubes immersed in a water bath at 37ºC. One test tube served as an experimental sample, and the other as control. 50 µl of serum was added to the experimental sample, which was incubated for 15 min at 37ºC. Next, 2 ml of a sodium azide solution was added to the experimental and control samples. 50 µl of serum was added to the control sample, and both samples were mixed. The absorbance of the experimental sample was measured at a wavelength of 540 nm, using the control sample as a blind test. Ceruloplasmin activity was determined with the use of the standard curve. The standard curve was plotted based on the optical density values for known ceruloplasmin concentrations. Total protein level. Whole blood samples were centrifuged for 5 min at 1,000 g to separate blood cells from the serum. 5 µl of serum was placed in the wells, and 25 µl of reagent A and 200 µl of reagent B were added (Rio-Rad, Hercules, USA). Well contents were gently stirred with a pipette. The microplates were incubated at room temperature for 15 min. Next optical density was measured in a microplate reader at 620 nm. Total protein level was determined using a standard curve as a reference. The standard curve was plotted based on optical density values for known protein dilutions. γ-globulin level. Whole blood samples were centrifuged for 5 min at 1,000 g to separate blood cells from the serum. The optical density of total protein was determined according to following procedure: 0.1 ml of serum was placed in the microplate wells, and 0.1 ml of 12% polyethylene glycol (10,000 kD) (Sigma Chemical Co.) suspended in deionised water was added. The microplates were incubated at room temperature for 2 h, and well contents were stirred continuously. The microplates were centrifuged for 10 min at 5,000 g to separate the γ-globulin fraction bound by polyethylene glycol (plate sediment) from the remaining total protein fraction, which constituted the supernatant. The optical density of supernatant was measured in a microplate reader at 620 nm. The optical density of supernatant was subtracted from the optical density of total protein. γglobulin content was determined using a standard curve (plotted earlier for total protein) as a reference, based on the ability of γ-globulins to bind with polyethylene glycol and precipitate. Evaluation of non-specific cellular immunity parameters. The metabolic activity of blood phagocytic cells was determined based on the intracellular measurements. Respiratory burst activity (RBA) after stimulation with PMA (Phorobol Myristate Acetate, Sigma), was determined according to the method described by Chung and Secombes (6) and adapted for dogs by Siwicki et al. (30). The isolated cells were resuspended in RPMI-1640 medium (Sigma) at 106 cells/mL. On 96-well U-shaped microplates, 100 µl of the isolated blood leukocytes was mixed with 100 µl of a 0.2% nitro blue tetrazolium (NBT, Sigma) solution in 0.2 M phosphate buffer (pH 7.2), and 1 µl of PMA at a concentration of 1 mg/mL in ethanol was added. After 30 min of incubation at 370C, the supernatant was removed from each well. The cell pellet was washed with absolute ethanol, and three times with 70% ethanol, and then was dried at room temperature. The amount of extracted reduced NBT after incubation with 2M KOH and DMSO (dimethylsulfoxide, Sigma) was measured colorimetrically at 620 nm in a microplate reader (Tecan Sunrise). All samples were tested in triplicate, and the results are presented as mean values. Potential killing activity (PKA) of mononuclear (MN) phagocytes and polymorphonuclear (PMN) phagocytes was determined in isolated blood leukocytes stimulated with killed microorganisms, according to the method presented by Rook et al. (26) and adapted for dogs by Siwicki et al. (30). On 96-well U-shaped microplates, 100 µl of leukocytes was mixed with 100 µl of 0.2 % NBT in phosphate buffer (pH 7.2), and 10 µl of killed Staphylococcus aureus strain 209P (containing 106 bacteria) was added. The mixture was incubated for 1 h at 370C and the supernatant was removed. The cell pellet was washed with absolute ethanol and three times with 70% ethanol, and then was dried at room temperature. This was followed by the 183 differences between groups was verified by the Student's t-test with the use of GraphPad Prism 5 software. addition of 2 M KOH and DMSO to each well. The amount of extracted reduced NBT was measured at 620 nm in a microplate reader (Tecan Sunrise). All samples were tested in triplicate, and the results are presented as mean values. Evaluation of specific cellular immunity parameters. Lymphocyte proliferation rates after stimulation with mitogens, concanavalin A (ConA), and lipopolysaccharide (LPS), were determined by MTT spectrophotometry (OD 570 nm) using (3-[4,5dimethylthiazol-2yl]-2,5-diphenyltetrazolium bromide – 3-[4,5-dimethyl-2-thiazol]-2,5-diphenyl-2H-tetrazolium bromide), as described by Mosmann (23). MTT (Sigma) was dissolved in PBS at a concentration of 5 mg/ml and filtered. On 96-well culture plates (Sarstetd, USA), 100 µl of blood lymphocytes in RPMI - 1640 containing 10% FCS, 2 mM L-glutamine, 0.02 mM 2-mercaptoethanol, 1% hepes buffer, and penicillin/streptomycin (100 U/100 µg/ml) was mixed with 100 µl of RPMI - 1640 containing mitogens ConA (5 µg/ml), PHA (10µg/ml) or LPS (20 µg/ml). After 72 h incubation at 370C in a 5% carbon dioxide atmosphere (Memmert Incubator), 50 µl of MTT solution was added into each well, and plates were incubated for 4 h at 370C. After incubation the plates were centrifuged (1,400 g, 150C, 5 min). Supernatants were removed and 100 µl of DMSO (Sigma) were added into each well and incubated for 15 min at room temperature. After incubation, the solubilised reduced MTT was measured colorimetrically at 570 nm in a microplate reader (Tecan Sunrise). All samples were tested in triplicate, and the results are presented as mean values. The final results are presented as the reactivity index (RI). Statistical analysis. The obtained results were processed statistically by a one-factorial analysis of variance in an orthogonal design. The significance of Results The obtained results indicate a significant effect of Biolex-MB40 on the analysed parameters of humoral and cellular immunity in lambs. An analysis of humoral immunity indicators– lysozyme and ceruloplasmin activities (Table 1) – revealed significantly (P≤0.01) higher values in the experimental group fed a diet with the addition of dried brewers’ yeast Saccharomyces cerevisiae than in the control group administered feed without yeast supplementation, over the entire experimental period. As regards the serum levels of γ-globulins (Table 1), their significant increase in the experimental group was reported only on the 30th (P≤0.05) and 60th (P≤0.01) d of the experiment in comparison with control group. In comparison with day 0 a significant increase in both ceruloplasmin and lysozyme activities (P≤0.01) was noted only in the experimental group on the successive days (15, 30, 60) of the experiment. Serum total protein content showed no significant differences between the experimental and control group (Table 1). In regard to the investigated indicators of nonspecific cellular immunity – RBA and PKA of phagocytes, and of specific cellular immunity - MTT stimulated with LPS and ConA (Table 2) – a statistically significant (P≤0.01 or P≤0.05) increase in their values was observed in the experimental group in comparison with the control group. The RBA in the group II was not marked by a significant increase in comparison with the group I only on the 60th d of the experiment. The RBA and MTT stimulated with LPS and ConA (Table 2) showed a significant increase (P≤0.01) only in the group I on successive days (15, 30, 60) of the experiment in comparison with day 0. Table 1 Effect of Biolex-MB40 on the parameters of non-specific humoral immunity in lambs Parameter Group 0 x Lysozyme activity (mg/L) Ceruloplasmin activity (mg/L) γ-globulin level (g/L) Total protein content (g/L) I II I II I II I II 0.79 0.79 31.75 31.20 30.22 34.11 57.18 57.10 15 SD 0.08 0.06 0.61 0.77 5.41 6.81 4.07 2.85 x B 0.79 1.09 A** 31.28 B 37.18 A** 33.17 36.44 62.28 61.67 Experimental day 30 SD x 0.09 0.77 B 0.09 1.14 A** 1.15 31.60 B 0.98 37.52 A** 2.83 31.61 b 5.82 35.44 a 3.88 57.30 2.24 56.17 60 SD 0.08 0.06 0.49 0.7 1.27 3.87 2.26 1.53 a, b - P≤0.05; A, B - P≤0.01; SD - standard deviation; I – experimental group; II – control group. * P≤0.05 in comparison with experimental day 0, ** P≤0.01 in comparison with experimental day 0. x B 0.81 1.17 A** 31.22 B 37.48 A** 30.67 B 39.44 A 58.15 56.25 SD 0.06 0.04 0.61 1.38 3.13 3.83 3.22 3.75 184 Table 2 Effect of Biolex-MB40 on the parameters of specific and non-specific cellular immunity in lambs Experimental day Parameter 0 Group x I RBA (OD 620 nm) II I PKA (OD 620 nm) II I MTT-ConA (RI) II I MTT-LPS (RI) II 0.51 0.51 0.47 0.50 1.15 1.20 1.10 1.12 15 SD 0.06 0.02 0.04 0.04 0.08 0.15 0.05 0.11 x SD 0.51 0.58 0.56 B A* 1.30 1.55 b a** 0.45 b a** 1.02 1.36 30 B A** 0.05 0.04 0.02 0.04 0.05 0.21 0.08 0.07 x 0.49 0.57 A** 0.45 0.54 B A** 0.90 1.32 B A 1.34 1.61 B B A** 60 SD x 0.05 0.52 0.02 0.03 0.03 0.08 0.19 0.06 0.09 SD 0.02 B 0.02 A 0.03 b 0.12 a** 0.03 B 0.06 A** 0.07 0.56 0.43 0.54 1.35 1.53 1.03 1.33 0.05 ** Symbols as in Table 1. Discussion The conducted study was the first ever attempt to determine the stimulating effect of a natural immunostimulator – Biolex-MB40 containing increased levels of β-1,3/1,6-D-glucan and MOS – on selected indicators of specific and non-specific humoral and cellular immunity in lambs. Glucans are a group of compounds known as glucose homopolymers. They are isolated from fungi, yeast, and plants, including oat and barley. Glucans derived from yeast and fungi have 1-3 bonds, and may occasionally feature additional 1-6 branches, whose number varies subject to glucan type. Glucans isolated from barley and oat are mostly linear compounds comprising regions with 1-4 bonds that separate smaller structures with 1-3 bonds (1). The most frequently described glucans that have been proven to have a stimulating effect on the immune system are: β-(1,3)(1,6)-glucan extracted from Saccharomyces cerevisiae, scleroglucan produced by Sclerotium glucanicum, grifolan (GRN) isolated from Grifola frondosa, SSG found in Sclerotinia sclerotiorum, and laminarin extracted from Laminaria digitata (2). The biological activity of β-glucan is determined by its origin, occurrence frequency, isolation method, size (molecular weight), physicochemical properties (such as solubility), primary structure, shape, degree of branching, and polymer charge (36). High molecular weight β-glucans (e.g. zymosan) may directly stimulate leukocytes, enhancing their phagocytic, cytotoxic, and antibacterial activity, including the production of reactive oxygen species and indirect nitrogen compounds. Low and medium molecular weight β-glucans (e.g. phosphate glucan) have a weaker effect on immune system cells. Short β-glucans molecules (e.g. laminarin with molecular weight of <5,000-10,000) are mostly inactive (4). Until recently, MOS were rarely investigated in regard to their effect on the immune system of animals. The conducted studies demonstrated that MOS contributed to a higher production of antibodies (28, 32), a drop in T cell number in peripheral blood (32, 24), a decrease in haptoglobin concentrations (5), and had no effect on the production of IL-6 (5). In this study, a significant increase in lysozyme and ceruloplasmin activities, and a rise in γ-globulin levels were observed in the group of experimental lambs fed diets with the addition of Biolex-MB40 in comparison with the control group. No such effect was noted as regards serum total protein (Table 1). Similar results were reported by other authors. Kokoshis et al. (14) demonstrated that after stimulation with β-glucan, lysozyme activity grew proportionally with phagocytosis, which was also enhanced by this compound. β-glucan binds to the receptors on the cell surface and activates transcription factors for plasma proteins. β-glucan and MOS also directly contribute to higher immunoglobulin levels and support the proliferation of B cells (7, 12, 16, 28, 32). By stimulating phagocytes to produce IL-1, IL-6, and TNFα, β-glucan affects the synthesis of acute phase proteins, including ceruloplasmin. Guzdek and Rokita (10) demonstrated a stimulating effect of curdlan sulphate (sulphate derivative of curdlan –1,3-β-glucan) on the levels of selected blood proteins, including ceruloplasmin. According to the above authors, the observed effect is caused mainly by the activation of transcription factors (mostly NF-κB) inducing protein synthesis. This observation validates the previous thesis. The results of other studies (22) also point to an increase in γ-globulin levels after the supplementation of lamb diets with β-1,3/1,6-D-glucan. Krakowski et al. (15) studied the immunostimulating effect of β-1,3/1,6D-glucan on pregnant mares and did not observe significant differences between the total protein content and γ-globulin levels in the offspring of mares fed 185 glucan. However, an increase in the total protein content and selected immunoglobulin levels in mare colostrum was observed. In this study, Biolex-MB40 also stimulated the proliferative activity of T and B cells. β-glucan and MOS bind with the C3R receptor on the surface of B and T cells to induce a cascade reaction and activate the NF-κB transcription factor. This factor induces the expression of cytokines, mostly IL-2 and IL-4, which stimulate the proliferation of B and T cells. NF-κB also exerts an indirect effect by activating phagocytes that remain in a mutual relationship with lymphocytes. The results of this study, relating to the stimulating effect of β-glucan and MOS on the proliferative activity of lymphocytes (Table 2), are consistent with the findings of other authors. Suzuki et al. (33) reported higher levels of proliferative activity of B cells (stimulated with LPS) and T cells (stimulated with ConA) in mice orally administered SSG (β-1,3-glucan isolated from Sclerotinia sclerotorium) for 5 and 10 consecutive days in the amount of 40 and 80 mg/kg/d. Similar results were reported by Szymańska-Czerwińska et al. (34) in a study of calves orally administered Alphamune (MOS and β-glucan) in daily doses of 14 g per animal. Zhao et al. (37) noted increased activity of lymphocytes stimulated with LPS and ConA following the administration of a different glucan type, FPS-1 (1,6-βglucan found in Aconitum carmichaeli). Mao et al. (20) studied the effect of β-glucan extracted from Astragalus membranaceus on lymphocyte proliferation in pigs twice immunised with LPS and in non-immunised pigs. The lymphocytes of both immunised and nonimmunised pigs receiving β-glucan were marked by higher proliferative activity stimulated by ConA. The above authors also demonstrated β-glucan's contribution to higher levels of IL-2 activity, thus confirming the previous observations that β-glucan activates lymphocytes through the stimulation of cytokine synthesis. In this study, the supplementation of lamb diets with Biolex-MB40 enhanced the activity of monocytes and granulocytes, leading to an increase in the RBA and PKA of phagocytes (Table 2). RBA and PKA tests evaluate changes in the oxygen metabolism of neutrophil granulocytes, known as the respiratory burst, and they are an indirect measure of the phagocytic activity of neutrophils. Nevertheless, the above tests are characterised by a different mechanism of neutrophil activation. In the RBA test, the switching of cell metabolism to glucose oxidation in the pentose cycle and the activation of the membrane enzyme complex – NADPH oxidase (nicotinamide adenine dinucleotide phosphate-oxidase) takes place with the involvement of phorbol ester (PMA) by the extra-receptor pathway, with the omission of signal transduction at the receptorNADPH oxidase level, and through direct stimulation (phosphorylation) of protein kinease C (PKC). Active NADPH oxidase continues to catalyse oxygen reduction to the superoxide anion radical O2-. As a substrate in various biochemical reactions, this radical supports the formation of other reactive oxygen species, including hydrogen peroxide, hydroxyl radical, singlet oxygen, and hyperchlorous acid. They have one or more lone electron pairs, which make them highly reactive. Found in phagolysosomes, they are toxic for bacteria, fungi, parasites, and neoplastic cells, and they determine the oxygen-dependent mechanism of microbial killing by phagocytes. Hyperchlorous acid and chloramine are the most potent antimicrobial oxidising agents produced by neutrophils. In the PKA test, the activation of the respiratory burst, as a result of the phosphorylation of cytoplasmic components of NADPH oxidase, starts from signal transmission at the ligand-receptor level, where the phagocyted strain of Staphylococcus aureus binds to the TLR2/TLR6 receptors of neutrophil granulocytes via the ligand modulin (PAMP – pathogen associated molecular patterns). Following the formation of superoxide radicals, the added NBT is reduced to insoluble formazan in the neutrophil cytoplasm. The exact mechanism of β-glucan's and MOS's impact on phagocyte functions has not been fully elucidated. β-glucans and MOS bind with the C3R complement receptor to activate phagocytes. By becoming attached to specific receptors on the cell surface, including the C3R complement receptor, lactosylceramide (CDw17), Dectin-1, and selected scavenger receptors (1, 3), β-glucan induces a reaction cascade, which activates the nuclear factor κB (NF-κB). NF-κB becomes attached to the promoter region of cytokines, such as IL-1, IL-6, and TNF-α, to induce their synthesis. It may also stimulate reactions, enhance the expression of iNOS (nitrogen oxide ligase), and other enzymes that participate in the processes of active elimination of microorganisms, such as the production of reactive oxygen species and lysozyme synthesis. The results noted by other authors concerning a stimulating effect of β-glucans on phagocyte activity in various animal species are consistent with the findings of this study involving the Biolex-MB40 supplement. Luhm et al. (18) demonstrated that NFκB induced higher levels of IL-8 and IL-1 synthesis in persons supplemented with 1,3-β-glucan. Similar results were noted by Kataoka et al. (13), who observed a strongly activating influence of linear glucan (curdlan) on NFκB. Linear β-glucans bind with phagocytes not only through the above receptors, but also through the TLR receptor (Toll-like receptors) and the TIR domain (similar to the domain in IL-1 receptors). Other studies (21) point to a stimulating effect of soluble glucan PGG (Betafectin®, β-1,3/1,6-D-glucan) on TNF-α and IL-1β synthesis with the involvement of monocytes and macrophages. An increase in superoxide anion production was also noted. The above authors found that PGG was able to stimulate the synthesis of the above compounds only when immobilised on the carrier. They argued that the above is explained by the fact that macrophage activation requires the cross-coupling of ligands with the receptor, as observed in molecular (insoluble) β-glucans, such as β-1,3/1,6-D-glucan investigated in this study. Sakurai et al. (27) demonstrated a stimulating effect of another soluble β1,3/1,6-D-glucan (SSG) on the production of TNF-α, nitrogen oxide, IL-1, and IL-6 by macrophages. The best results were noted during co-stimulation with INF-γ, 186 which testifies to a weaker effect of soluble glucans. Several years earlier, Hamuro et al. (11) confirmed the positive effects of eight types of 1,3-β-glucans (lentinan isolated from Lentinus edodes, pachyman extracted from Poria cocos and its chemically modified derivatives) on the cytotoxicity of macrophages. The above authors also suggested another possible mechanism of phagocyte activation. They argued that the studied β-glucans were capable of activating the complement receptor in an alternative manner, i.e. by activating its components, in particular C3b. The activated complement receptor stimulates the potential killing activity of phagocytes, increasing their ability to eliminate pathogens. 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