Enzyme and Microbial Technology 55 (2014) 113–120

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Kocuran, an exopolysaccharide isolated from Kocuria rosea strain BS-1 and evaluation of its in vitro immunosuppression activities C. Ganesh Kumar a,b,∗ , Pombala Sujitha a,b a b

Academy of Scientific and Innovative Research, CSIR-Indian Institute of Chemical Technology, Uppal Road, Hyderabad 500007, India Medicinal Chemistry and Pharmacology Division, CSIR-Indian Institute of Chemical Technology, Uppal Road, Hyderabad 500007, India

a r t i c l e

i n f o

Article history: Received 20 August 2013 Received in revised form 18 October 2013 Accepted 21 October 2013 Keywords: Kocuran Kocuria Antioxidant Immunosuppression Macrophages

a b s t r a c t In an ongoing survey for bioactive potential of microorganisms from different biosphere zones of India, a promising Kocuria rosea strain BS-1 was identified which produced an exopolysaccharide (designated as Kocuran) exhibiting in vitro antioxidant and immunosuppression properties. Kocuran was characterized as a heteropolysaccharide with repeating monosaccharide residues of glucose, galactose, mannose and glucuronic acid with an average molecular mass of 51.2 kDa. In RAW 264.7 macrophages, Kocuran significantly downregulated the LPS-stimulated ROS, NO, TNF-␣, IL-6 and C3 complement component secretion to 4.71 ± 0.08%, 4.11 ± 0.06%, 11.19 ± 0.06 pg ml−1 , 9.12 ± 0.07 pg ml−1 and 20.81 ± 0.06 ng/106 cells ml−1 , respectively. Furthermore, it inhibited the PHA-stimulated proliferation of human peripheral blood mononuclear cells with IC50 of 100.13 ± 2.1 ␮g ml−1 . In addition, the classical and alternative pathway mediated hemolysis was also inhibited with CH50 and AH50 of 100.96 ± 1.75 and 98.60 ± 1.93 ␮g ml−1 , respectively. Kocuran did not inhibit the LPS-induced LAL enzyme and the binding of FITC-LPS to macrophages suggesting that Kocuran does not neutralize the LPS activity. These results demonstrate the in vitro suppression of activation and macrophage-derived inflammatory cytokines and complement mediated hemolysis indicating its in vitro immunosuppression activity. © 2013 Elsevier Inc. All rights reserved.

1. Introduction In the recent years there is an increased focus to search for novel natural products or microorganisms of economic importance from the global biodiversity. Over the last few decades, novel microorganisms isolated from diverse geographical locations and niches across the world are explored as promising resources of novel drug candidates for development of new drugs by the pharmaceutical companies [1]. Several bacteria, fungi, algae, lichens and plants are known to produce natural metabolites that can alter the immune response. Among these metabolites, the polysaccharides are reported as a good candidate for modulating immune system by activating or suppressing the immune response [2]. Bacteria are known to produce heterogeneous species specific extracellular polysaccharides (EPS) with different structural complexities [3]. These EPS are reported to exhibit potential biological activities such as immunomodulating, anti-tumor, antiviral, anti inflammation and antioxidant properties [2,4,5].

∗ Corresponding author at: Medicinal Chemistry and Pharmacology Division, CSIR-Indian Institute of Chemical Technology, Uppal Road, Hyderabad 500007, India. Tel.: +91 40 27193105; fax: +91 40 27193189. E-mail address: [email protected] (C.G. Kumar). 0141-0229/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.enzmictec.2013.10.007

The modulation of immune responses is regulated by suppressing or stimulating the release of ROS, RNS and proinflammatory cytokines from macrophages and immune regulatory cells [6]. In the immune system, macrophages are the primary defense cells that initiate immune responses and activate immune cells like lymphocytes NK cells and dendritic cells. The activated macrophages express increased levels of class II MHC molecules and costimulatory molecules that trigger the immune response serve as effector cells. Furthermore, macrophages are unique components of the innate immunity and have a range of functions related to the activation process. Activated macrophages become more efficient antigen presenting cells because they express increased levels of class II MHC molecules and co-stimulatory molecules [7]. Some of the bacterial EPS produced by Pseudomonas aeruginosa [8] and Lactobacillus rhamnosus RW-9595M [9] have been reported to exhibit immunosuppressive properties by suppressing the release of macrophage-derived regulatory molecules. There is a paucity of information on EPS producers from genus Kocuria except strains of Kocuria erythromyxa 32f and Kocuria rosea 32d which produced EPS with biosurfactant property [10], and Kocuria turfanesis strain-J which produced a biosurfactant that emulsified pesticides at extreme environmental conditions [11]. However, the EPS and biosurfactant produced by these Kocuria strains have not been characterized. In continuation to our exploration for bacterial EPS from a biotechnological perspective [12,13],

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and also in view of the importance of microbial EPS as antioxidants and immunomodulating agents, different microbial strains isolated from different biosphere zones of India and maintained in the in-house culture collection were screened for EPS production. Strain BS-1 was earlier isolated in our laboratory from a petroleum contaminated soil sample and deposited in the in-house culture collection as accession number ICTB1799. The strain BS-1 produced an EPS with biosurfactant property which was identified as K. rosea based on morphological and cultural characteristics. The 16S rDNA gene sequence is deposited in GenBank with accession number HQ218995 [14]. In the present study, the EPS produced by K. rosea strain BS-1 was characterized and its in vitro immunosuppression activities were evaluated in RAW 264.7 mouse macrophages and human peripheral blood mononuclear cells (PBMC).

The average molecular mass of Kocuran was determined by gel permeation chromatography (Agilent 1100 Series HPLC system, TOSOH Corporation, Japan) equipped with a RID and a TSK G5000PWXL gel column (7.8 mm × 300 mm) and a TSK PWXL (6.0 mm × 40 mm) guard column using dextran standards ranging from 10,680 to 578,500 Da. The Fourier transform infrared spectrum (FT-IR) was recorded on the Thermo-Nicolet Nexus 670 FT-IR spectrophotometer (ThermoFisher Scientific Inc., Madison, WI, USA) using KBr pellets at a resolution of 4 cm−1 in the wavenumber region of 400–4000 cm−1 . The cross-polarization/magic angle spinning (CP/MAS) 13 C NMR experiments were performed on a Varian Unity Innova spectrometer operating at 400 MHz for 1 H and 100 MHz for 13 C at room temperature. The EPS samples used for solid-state NMR analysis were gently grinded to ensure sample homogeneity and were packed in a zirconium oxide rotor, sealed with a Kel-f cap. The CP time was 4 ms and the rotor spinning frequency was 10 kHz. The 1 H and 13 C pulse widths were 2.9 and 3.5 ␮s, respectively, with a repetition time of 4 s. The spectral width was 33,183.3 Hz with an accumulation of 512 scans. The data was processed with 8192 data points (Fourier number) with an exponential line broadening of 30 Hz. 2.4. Analysis of monosaccharide composition of Kocuran

2. Materials and methods 2.1. Materials All the reagents used in the study such as dextran standards (ranging from 10,680 to 578,500 Da), 1,1-diphenyl-2-picrylhydrazyl (DPPH), phenazine methosulfate (PMS), nicotinamide adenine dinucleotide (NADH), Nitroblue tetrazolium (NBT), Dulbecco modified Eagle medium (DMEM), HEPES, penicillin, streptomycin, anti-mycotic solution, fetal bovine serum (FBS), sodium pyruvate, RPMI-1640, Trypan blue, gelatin veronal buffer (GVB), phosphate buffered saline, trypsin EDTA, phytohemagglutinin (PHA), 2,7-dichlorofluorescein diacetate (DCFH-DA), lipopolysaccharide (LPS), fluorescein isothiocyanate (FITC)-labeled LPS, tertiary butyl peroxide (t-BHP), S-nitroso-N-acetyl-d,l-penicillamine (SNAP), Polymyxin B, interferon-␥, sheep erythrocytes sensitized with anti-sheep antibody, 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), actinomycin D, dimethyl sulfoxide, rabbit erythrocytes, ethylene glycol-bis[,3-aminoethyl ether]N,N,N1 ,N1 -tetraacetic acid (EGTA) were procured from Sigma–Aldrich, St. Louis, MO, USA. TNF-␣ and IL-6 ELISA kits were obtained from Biosource International Camarillo, CA, USA. Affi-Prep Polymyxin Matrix column was procured from BioRad, Hercules, CA, USA. Chromogenic Limulus Amoebocyte Lysate (LAL) assay kit was procured from Lonza Inc., Allendale, NJ, USA. 2.2. Culture conditions and EPS production

The purified Kocuran was trimethylsilylated [18] to determine the monosaccharide composition by GC and compared with standard monosaccharides. The EPS was also carboxyl-reduced [19] and degraded with lithium-ethylenediamine [20]. The native, carboxyl reduced and lithium-ethylenediamine degraded EPS products were converted to partially methylated alditol acetates [21] and analyzed by GC–MS (Waters Corp., Milford, MA, USA) equipped with an HP-5MS column (Agilent Technologies, Wilmington, DE, USA) using a temperature program of 120–180 ◦ C, ramped at 5 ◦ C/min and 180–250 ◦ C, ramped at 2 ◦ C/min, and the mass conditions were: ionization mode with EI, ionization energy of 70 eV, a current intensity of 500 ␮A, and ion source temperature at 250 ◦ C. The partially methylated alditol acetates were analyzed relative to 1,5-di-O-acetyl-2,3,4,6-tetra-O-methylglucitol. Further, the anomeric configuration was determined from the chromium trioxide oxidation of native and carboxyl reduced EPS [22] using myo-inositol as an internal standard. 2.5. Antioxidant activities The DPPH assay [23] was performed using different concentrations of Kocuran incubated with methanolic solution of 160 ␮M DPPH at 35 ◦ C for 30 min in dark. The absorbance of the DPPH solution was measured at 517 nm. The inhibitory effect on scavenging superoxide anions (O2− ) was monitored by PMS and NADH systems [24]. In brief, Kocuran at different concentrations (10, 20, 40, 80 and 200 ␮g ml−1 ) were added to the reaction mixture containing 3 ml of 16 mM Tris–HCl buffer (pH 8.0), 78 mM NADH, 50 ␮M NBT and 10 ␮M PMS, then incubated at 37 ◦ C for 5 min and the absorbance was measured at 560 nm. The lipid peroxidation [25] and reducing power assay [26] experiments were conducted in triplicates using ascorbic acid as standard. The inhibitory effect on the generation of free radicals was calculated as follows:

Growth kinetics profile of Kocuria strain BS-1 with reference to EPS production was assessed in minimal salts medium (MSM, pH 8) containing (per liter): 10 g glucose, 2 g sodium nitrate, 0.5 g NH4 SO4 , 2.5 g KH2 PO4 , 2.0 g K2 HPO4 , 0.2 g MgSO4 and 1 g NaCl and incubated at 35 ◦ C with agitation at 180 rpm for 96 h in an Ecotron (Infors AG, Switzerland) rotary shaker. Samples (10 ml) were withdrawn at a periodic interval of 12 h, centrifuged (Sorvall RC5 C Plus, Kendro Lab Products, Ashville, NC, USA) at 10,000 rpm for 20 min and the cell biomass was determined on cell dry weight basis. The bacterial growth was determined by optical density (OD) measurement of the diluted culture using a UV–visible double beam spectrophotometer (Lambda 25, Perkin-Elmer, Shelton, CT, USA) at 600 nm and expressed as mg dry cell weight ml−1 . The dry weight of cells per ml at corresponding OD was obtained and standard curve for OD versus dry weight was plotted. The absorbance of 600 nm equal to 1.0 corresponded to 0.63 mg dry cell weight ml−1 was experimentally determined for this bacterial strain. The EPS was precipitated with two volumes of ice-cold isopropanol. The precipitate was recovered by centrifugation at 10,000 rpm for 20 min and was dialyzed (MWCO 6000–8000 Da) against deionized water for 24 h at 4 ◦ C and then vacuum dried to obtain the crude EPS and the yield was determined on dry weight basis. The growth kinetics profile was plotted with reference to EPS production and bacterial growth as a function of time.

The RAW 264.7 mouse macrophages (ATCC No. CRL-2278) were cultured in DMEM supplemented with 10% FBS containing 100 ␮g ml−1 each of penicillin and streptomycin and incubated at 37 ◦ C in a 5% CO2 humidified incubator. Human peripheral blood mononuclear cells (PBMC) isolated from normal human donors (HIV-1, HIV-2, hepatitis and bacteria-free) were purchased from Lonza Inc., Allendale, NJ, USA. The cells were cultured in RPMI-1640 containing glutamine (2 mM), 10% FBS, penicillin (50 ␮g ml−1 ), streptomycin (50 ␮g ml−1 ), and anti-mycotic solution (0.1 mM) and incubated at 37 ◦ C in 5% CO2 humidified incubator.

2.3. Purification and characterization of EPS

2.7. Measurement of intracellular ROS and NO in RAW 264.7 macrophages

The bacterial strain BS-1 was cultured in the MSM liquid medium as described above. The medium was centrifuged at 8000 rpm at 4 ◦ C to obtain the cell-free supernatant, which was extracted with ice cold isopropanol, centrifuged at 10,000 rpm for 20 min and filtered through 0.45 ␮m Whatman filter paper. The extract was dialyzed (MWCO 6000–8000 Da) against deionized water for 48 h at 4 ◦ C. The EPS was initially treated with DNase, RNase, and Proteinase K and the endotoxin contaminants were removed using Affi-Prep Polymyxin Matrix. The EPS was further purified on DEAE cellulose (Cl− ) column (2.5 cm × 50 cm) and eluted with 0.2 M NaCl. The EPS fractions free of contaminants obtained were pooled, concentrated, dialyzed and then lyophilized. The purified EPS was designated as Kocuran. Later, it was acid hydrolyzed (water, trifluoroacetic acid and acetic acid, 75:5:20, v/v), heated to 120 ◦ C for 6 h and vacuum evaporated followed by deacetylation with trifluoroacetic acid and water (10:90, v/v). The hydrolyzed product was subjected to colorimetric analysis for sugars [16], proteins [17] and uronic acids [15] using appropriate standards.

The intracellular ROS was measured using DCFH-DA [27] which is oxidized to the fluorescent compound, 2,7-dichloro fluorescein (DCF) by intracellular ROS. RAW264.7 cells (2 × 106 cells well−1 in 24-well plate) were treated with Kocuran at concentrations of 1, 10, 20 and 50 ␮g ml−1 and then stimulated with 100 ng ml−1 of LPS for 24 h. The intracellular ROS was measured by recording the absorbance at 485 nm excitation and 520 nm emission wavelengths using a fluorescence spectrophotometer (Model F4500, Hitachi High Technologies Corp., Tokyo, Japan). The inhibitory effect of Kocuran on the generation of intracellular ROS was also measured using tertiary butyl peroxide (200 ␮M, t-BHP), a known ROS generator. The amount of nitric oxide (NO) released into the culture supernatants after the LPS treatment in the presence and absence of Kocuran was quantified using Griess reagent [28]. The inhibitory effect of Kocuran on NO production was also studied by treating the macrophages with S-nitroso-N-acetyl-d,l-penicillamine (100 ␮M), a known NO generator. All the results are reported as the means ± S.D. of three independent experiments.

Inhibitory effect(%) = Acontrol − Asample /Acontrol × 100.

2.6. Cell culture

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2.8. Analysis of C3, TNF-˛ and IL-6

The in vitro proliferation activity of Kocuran on PBMC was assessed using MTT method [29]. Briefly, PBMC were cultured in 96-well plates overnight, stimulated with PHA (5 ␮g ml−1 ) and then treated with different concentrations of Kocuran (1–200 ␮g ml−1 ) for 48 and 72 h. Finally, 20 ␮l of MTT (5 mg ml−1 ) was added to each well and incubated for 4 h and then the formazan crystals formed were dissolved in 150 ␮l well−1 of DMSO. The absorbance was measured at 570 nm in a microplate reader (BioRad Laboratories, Hercules, CA, USA). Further, the in vitro cytotoxicity was determined from the total and viable cell numbers of PBMC counted after trypan blue staining and the percentage of viable cells was calculated as follows: Viability (%) = Viable cell number/Total cell number × 100.

-1

0.6

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0.5 EPS

Biomass

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0.2 0.5 0.1 0

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2.9. Lymphocyte proliferation assay

3

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Kocuran (mg ml )

The culture supernatants collected from the LPS-stimulated RAW 264.7 macrophages pretreated with Kocuran (1, 10, 20 and 50 ␮g ml−1 ) were determined for C3, TNF-␣ and IL-6 production using ELISA kits as per the manufacturer’s instructions.

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0 0

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Incubation (h) Fig. 1. Growth kinetics profile as a function of time for EPS production from Kocuria rosea strain BS-1. Values represent the means ± S.D. from three independent experiments.

3. Results and discussion 2.10. Complement mediated hemolytic assay The complement mediated hemolytic assay was performed using sheep erythrocytes sensitized with anti-sheep antibody where 100 ␮l of erythrocytes were mixed with 50 ␮l of human serum (1:500 diluted in 0.1 M HEPES buffer) in a 96 well plate. Kocuran at various concentrations (1, 10, 100 and 200 ␮g ml−1 ) was added to above mixture and incubated for 60 min at 37 ◦ C. In another set of experiments, rabbit erythrocytes (4.5 × 106 cells/ml suspended in GVB) were mixed with 8 mM EGTA, 2 mM Mg2+ and human serum (1:24 dilution) to initiate alternative pathway mediated hemolysis [30]. The samples were incubated in the presence of different concentration of Kocuran for 1 h at 37 ◦ C. After incubation, the samples were centrifuged at 10,000 rpm for 10 min and the absorbance of the supernatant was measured at 410 nm. The hemolytic inhibition fraction was calculated from (Amax − Asample )/Amax where Amax (maximal hemolysis) and Asample represents the absorbance of the sample in presence of human serum only and the sample along with Kocuran, respectively. All the experiments were performed in triplicate and the results are reported as the concentration of compound required for 50% inhibition of classical pathway mediated (CH50 ) and alternative pathway mediated (AH50 ) hemolysis.

2.11. Limulus amoebocyte lysate assay (LAL assay) The binding and neutralization of LPS by Kocuran was assessed using a quantitative chromogenic Limulus Amoebocyte Lysate (LAL) assay kit. Kocuran at various concentrations (1, 10, 100 and 200 ␮g ml−1 prepared in the pyrogen-free water) were incubated with LPS (1 ␮g ml−1 ) in a flat bottom non-pyrogenic 96-well plate at 37 ◦ C for 30 min to allow the binding of Kocuran to LPS. To this mixture, 50 ␮l of LAL reagent was added and incubated for 10 min and then 100 ␮l of LAL chromogenic substrate (Ac-Ile-Ala-Arg-p-nitroaniline) was added. The reaction mixture was incubated for 15 min and the reaction was terminated by adding 25% acetic acid. The yellow color formed due to the cleavage of the substrate was measured spectrophotometrically at 410 nm. Polymyxin B (10 ␮g ml−1 ) was used as positive control and all the results are reported as the means ± S.D. of three independent experiments and the decrease in the absorbance is due to the interaction of Kocuran with LPS and simultaneous inhibition of its activity.

3.1. EPS production as a function of time From the growth kinetic analysis, it was observed that EPS production was growth associated and the EPS production correlated with the cell growth (Fig. 1). The growth of K. rosea strain BS-1 was rapid up to 48 h at which stationary phase was reached. At 48 h, maximum EPS production (0.731 mg ml−1 ) was observed that remained constant until 96 h. The growth assessment with respect to EPS production as a function of time indicated that K. rosea strain BS-1 was a prominent EPS producer. 3.2. Purification and characterization of EPS Based on the calorimetric analysis, it was observed that the Kocuran EPS consisted of 91% sugars and 9% uronic acid content. This EPS is acidic in nature with an average molecular mass of 51,200 Da as revealed from gel permeation chromatography (Fig. 2). FT-IR spectrum revealed characteristic absorption peaks such as  = 3429, 2924.5, 1073 and 693.39 cm−1 corresponding to OH group, C H, C O and anomeric regions (Supplementary Fig. S1). Further the 13 C CP-MAS NMR (400 MHz) spectrum (Supplementary Fig. S2) revealed the presence of anomeric carbon signals at ı = 99.94 ppm. The chemical shifts at ı = 173.94 ppm, ı = 62.261 ppm corresponded to the carbonyl signals of carboxyl carbon (COO) and C6 carbon signals ( C6 ), respectively. The peak at ı = 71.267 can be due to the side spinning band and the peaks at ı = 32.996 ppm and ı = 14.33 ppm

2.12. FITC-LPS binding to RAW 264.7 macrophages The effect of Kocuran on LPS binding to RAW macrophages was demonstrated by incubating Kocuran at different concentrations (10, 100 and 200 ␮g ml−1 ) and Polymyxin B (10 ␮g ml−1 ) with fluorescein isothiocyanate (FITC)-labeled LPS (in an equal ratio) for 1 h at 4 ◦ C. Later, the RAW macrophages (2 × 106 cells well−1 in 24well plate) were treated with this LPS-Kocuran mixture and further incubated for 30 min at 37 ◦ C. The cells were extensively washed with ice-chilled PBS to remove the unbound LPS from the samples. The binding of FITC-LPS in the presence of Kocuran was monitored by measuring the fluorescence in each sample using FACS Caliber flow cytometer (BD Biosciences, San Jose, CA, USA).

2.13. Statistical analysis Statistical analysis was performed using GraphPad PRISM software version 3.0 (GraphPad Software, Inc, La Jolla, CA, USA). Data were expressed as the mean ± S.D. The unpaired Student t-test was adopted to determine the statistical significance. In all the comparisons, p < 0.05 was considered statistically significant.

Fig. 2. Gel permeation chromatogram of Kocuran from Kocuria rosea BS-1. The diagonal line in the figure represents the average molecular weight of the Kocuran polysaccharide. The average molecular weight of Kocuran was determined from the GPC analysis using dextran as internal standard.

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Fig. 3. Predicted structure of Kocuran produced by Kocuria rosea strain BS-1.

were attributed to methylene carbons ( CH2 ) and methyl carbons ( CH3 ), respectively. The GC analysis of alditol acetates from the native EPS showed the presence of d-glucose, d-galactose and d-mannose in the molar ratios of 1.5:0.7:0.1, while the carboxyl reduced EPS showed the presence of d-glucose, d-galactose and d-mannose with the molar ratios of 1.9:0.7:0.1 (Supplementary Table S1 and Supplementary Figs. S3 and S4). The observed increase in the molar ratio of glucose in the case of carboxyl reduced EPS indicated that the uronic acid present in EPS is glucuronic acid which was converted into glucose derivative upon carboxyl reduction. The results suggested that Kocuran is an acidic heteropolysaccharide composed of glucopyranosyl, galactopyranosyl, mannopyranosyl and glucopyranosyl uronic acid residues. The analysis of partially methylated alditol acetate (PMAA) derivatives of lithium-ethylenediamine degradation of native Kocuran (Supplementary Table S2 and Supplementary Fig. S5) showed the presence of 2,3,6-tri-O-methyl-1,4,5-tri-O-acetyl-dglucitol, 2,6-di-O-methyl-1,3,4,5-tetra-O-acetyl-d-galactocitol and 1,3,4,6-tetra-O-methyl-2,5-di-O-acetyl-d-mannitol derivatives in the ratio of 1.5:0.7:0.2. These three residues indicated the presence of 1,4-linked glucopyranosyl, 1,3,4-linked galactopyranosyl and 2-linked mannopyranosyl residues. It is envisaged that the mannopyranosyl is linked to galactopyranosyl through 2,3 linkages. Further, the increase in the molar ratio of 2,3,6-tri-O-methyl1,4,5-tri-O-acetyl-d-glucitol residue in case of carboxyl reduced EPS, indicated the presence of glucopyranosyl uronic acid residue through 1,4-linkage (Supplementary Fig. S6). Further, the ready oxidation of sugar residues analyzed after chromium trioxide oxidation indicated that the sugar residues have ␤-glycosidic linkages. The analysis of alditol acetates after chromium trioxide oxidation showed a rapid decrease in the amount of d-glucose, d-galactose and d-mannose residues (Supplementary Table S3) and a similar rapid decrease in the amount of d-glucose was observed with carboxyl reduced EPS, suggesting that all the sugar residues are in ␤-configuration. It was earlier observed that the rapid decrease in the amount of sugar residue is due to the abstraction of an axially oriented C-1 proton to give 5-heulosonic acid which results in the rapid disappearance of sugar residues with ␤-glycosidic linkage [22]. Based on the above analysis of the purified EPS, the tentative predicted structure of the repeating unit of Kocuran is shown in Fig. 3. 3.3. Antioxidant activities of Kocuran Kocuran scavenged DPPH free radicals, superoxide anions and lipid peroxyl radicals in a dose-dependent manner (Fig. 4). DPPH is an oxidizing radical that forms a stable free radical which is reduced and stabilized by antioxidants. The DPPH free radical inhibition values recorded for Kocuran are 23.56 ± 0.04%, 38.97 ± 0.06% and 62.98 ± 0.07% at 20, 40 and 80 ␮g ml−1 with the EC50 value of 51.28 ± 0.02 ␮g ml−1 whereas the EC50 value of ascorbic acid was 40.28 ± 0.05 ␮g ml−1 . The scavenging activity of DPPH free radicals by Kocuran can be attributed to the presence of reducing sugars such as glucose, galactose and mannose [31]. Further, the superoxide anion inhibition observed was 68.07 ± 0.04% at 80 ␮g ml−1 of Kocuran and the EC50 values were 42.78 ± 0.04 and 21.1 ± 0.03 ␮g ml−1 for Kocuran and ascorbic acid, respectively. Superoxide anions exert deleterious effects on biological system

Fig. 4. Antioxidant activities of Kocuran produced by Kocuria rosea strain BS-1. Antioxidant activities of Kocuran were assessed by measuring the scavenging ability of DPPH free radicals, inhibition of superoxide anion and lipid peroxidation, and reducing power. The results are represented as the percent activity and error bars represent the means ± S.D from three independent experiments performed.

by forming singlet oxygen and hydroxyl radicals upon decomposition. The superoxide anion scavenging by Kocuran can be attributed to the presence of free hydroxyl group which neutralizes the superoxide anions [32]. Similarly, Kocuran stabilized the lipid peroxidation product, malondialdehyde (MDA) and exerted inhibitory effect on lipid peroxidation with EC50 values of 117.64 ± 0.37 and 130.97 ± 0.54 ␮g ml−1 for Kocuran and ascorbic acid, respectively. In biological systems, MDA acts as a mutagen and exerts its deleterious effects by reacting with DNA bases to form DNA–DNA interstrand crosslinks or DNA-protein crosslinks [33,34]. These results demonstrated that Kocuran possess antioxidant capacity and is able to scavenge DPPH free radicals, superoxide anions and lipid peroxyl radicals. 3.4. Inhibition of ROS and NO generation in RAW 264.7 macrophages Murine RAW 264.7 macrophages are reported to show prototypical macrophage response when stimulated with classical stimulators like LPS or IFN-␥ [35]. The stimulated macrophages secretes ROS, NO, and proinflammatory cytokines such as TNF-␣, IL-1␤ and IL-6. Among the biomarkers associated with macrophage activation the ROS acts as secondary signaling molecules and regulate the inflammatory and immune responses associated with activated macrophages [36]. In addition, NO regulates physiological responses such as neurotransmission, blood pressure regulation, defense mechanisms and immune regulation and also exerts some deleterious effects like DNA fragmentation, destruction of mitochondrial membrane potential and inactivation of iron sulphur cluster containing enzymes [37]. Kocuran inhibited the LPS (100 ng ml−1 ) stimulated production of ROS and NO from RAW 264.7 macrophages in a dose-dependent manner (Fig. 5). After 24 h of treatment with 20 ␮g ml−1 of Kocuran, the percentage of ROS produced in LPS-stimulated macrophages was inhibited to 4.71 ± 0.08%, whereas the ROS production in the LPStreated and normal cells (treated with only DMEM medium) were 79.95 ± 0.12% and 4.01 ± 0.08%, respectively. Similarly, when RAW 264.7 macrophages were treated with 20 ␮g ml−1 of Kocuran, the LPS-stimulated NO level (66.89 ± 0.15%) considerably decreased to 4.11 ± 0.06% corresponding to the normal levels (4.17 ± 0.06%). Furthermore, MTT measurements demonstrated no loss of viability when RAW 264.7 cells were treated with Kocuran at various concentrations (Supplementary Fig. S7) indicating that the inhibition of ROS and NO production from LPS-stimulated RAW 264.7 macrophages was not associated with the cell death due to Kocuran

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Fig. 5. ROS and NO released in the presence of Kocuran in LPS-stimulated RAW 264.7 mouse macrophages. The RAW 264.7 macrophages were stimulated with LPS (100 ng ml−1 ) in the presence or absence of Kocuran at different concentrations (1, 10, 20 and 50 ␮g ml−1 ) for 24 h. The intracellular ROS and NO released were quantified using DCFH-DA protocol and Griess reagent, respectively. The normal cells represent the unstimulated group that was not treated with either LPS or Kocuran. The error bars represent as the means ± S.D. from three independent experiments performed. p < 0.05 was considered to be statistically significant.

treatment. Kocuran also inhibited the IFN-␥ stimulated ROS and NO production in RAW macrophages (Supplementary Fig. S8). In addition, Kocuran also inhibited the production of t-BHP induced ROS generation (Supplementary Fig. S9) and SNAP induced NO production (Supplementary Fig. S10) in RAW macrophages. These results demonstrated the efficacy of Kocuran to scavenge ROS and NO production in RAW macrophages. 3.5. Inhibition of TNF-˛, IL-6 and C3 formation in RAW 264.7 macrophages The stimulation of macrophages with LPS exerts endotoxin shock which induces the excessive secretion of inflammatory cytokines like TNF-␣, IL-1␤ and IL-6. These macrophage-derived cytokines trigger the generation of ROS in non-phagocytic cells; induce NO synthase and serine proteases, causing membrane permeabilization and DNA strand breaks [38]. In addition, they induce the expression of major histocompatibility complex, IFN␥ and complement components from different immune cells including T and B lymphocytes and also initiate a number of immune-cascade pathways [39]. In the present study, Kocuran was observed to suppress the secretion of macrophage-derived cytokines, TNF-␣ and IL-6, as well as C3 complement from LPS-stimulated RAW 264.7 macrophages in a dose-dependent manner after 24 h (Fig. 6a and b). In RAW 264.7 macrophages, the LPS treatment stimulated the TNF-␣ and IL-6 production levels to 215.01 ± 0.12 and 191.54 ± 0.15 pg ml−1 , respectively, while the normal macrophages (treated with DMEM medium) showed decreased TNF-␣ and IL-6 production levels of 8.15 ± 0.05 and 9.13 ± 0.05 pg ml−1 , respectively. Upon treatment with Kocuran at 50 ␮g ml−1 for 24 h, the TNF-␣ and IL-6 production levels from LPS-stimulated macrophages were suppressed to 11.19 ± 0.06 and 9.12 ± 0.07 pg ml−1 , respectively (Fig. 6a). Based on the in vitro data evaluation, it was suggested that Kocuran suppressed production of ROS, NO, TNF-␣ and IL-6 in macrophages. In addition, Kocuran remarkably inhibited the secretion of C3 complement component from the LPS-stimulated RAW 264.7 macrophages. C3 complement component is the central complement component involved in complement activation through classical, alternative and mannose-binding lectin pathways [40]. It is involved in several

Fig. 6. Inhibition of (a) proinflammatory cytokines TNF-␣ and IL-6, and (b) C3 complement component production by Kocuran in RAW 264.7 mouse macrophages. The RAW 264.7 macrophages were stimulated with LPS (100 ng ml−1 ) in the presence or absence of Kocuran at different concentrations (1, 10, 20 and 50 ␮g ml−1 ) for 24 h. The release of proinflammatory cytokines such as TNF-␣ and IL-6 and C3 complement component was measured using ELISA. The normal cells represent the unstimulated group that was not treated with either LPS or Kocuran. The error bars represent as the means ± S.D from three independent experiments performed. p < 0.05 was considered to be statistically significant.

pathophysiological conditions by producing anaphylotoxins such as C3a, C4a and C5a and forming membrane attack complex (MAC) causing lysis [41]. Hence the inhibition of upregulated C3 complement secretion is of primary concern to regulate the immune response and to treat various immune diseases such as autoimmune diseases, arthritis, etc. [40]. The LPS treatment increased the C3 complement component secretion from the normal unstimulated level of 1.81 ± 0.01 ng/106 cells ml−1 to a stimulated level of 55.89 ± 0.06 ng/106 cells ml−1 . When treated with Kocuran at 10, 20 and 50 ␮g ml−1 , the C3 complement component secretion levels in the LPS-stimulated macrophages were drastically reduced to 44.37 ± 0.07, 20.81 ± 0.06 and 2.05 ± 0.01 ng/106 cells ml−1 , respectively (Fig. 6b). The observed inhibitory effect on C3 complement secretion can be due to the downregulation of oxidative stress-induced redox signaling that activates the transcription factor, NF-␬B/AP-1, that induces the expression of several proinflammatory genes including TNF-␣, IL-6 and C3 complement component [42]. 3.6. Inhibition of human PBMC proliferation The macrophage-derived cytokines, TNF-␣, IL-2 and IL-6, acts as growth factors and activates the proliferation of immune cells such as T and B lymphocytes, NK cells and neutrophils [43]. The human

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Fig. 7. Cell proliferation assay of Kocuran on human PBMC. The human PBMC were incubated with PHA (5 ␮g ml−1 ) in the presence or absence of Kocuran at different concentrations (1, 10, 20 and 50 ␮g ml−1 ) for two periodic intervals, 48 and 72 h. The in vitro proliferation activity of Kocuran on human PBMC was assessed using the MTT assay. The normal cells represent the unstimulated group that was not treated with either PHA or Kocuran, while actinomycin D (1 ␮g ml−1 ) was used as positive control. The error bars represent as the means ± S.D from three independent experiments performed. p < 0.05 was considered to be statistically significant.

PBMC plays an important role in exerting immune responses through the activation and clonal expansion process [44]. The PBMC are known to be activated by PHA, a mitogen that stimulates the proliferation by interacting with the N-acetylgalactosamine glycoprotein present on the cells [45]. In the present study, Kocuran inhibited PHA-stimulated PBMC proliferation in a concentrationdependent manner (Fig. 7). At 100 ␮g ml−1 , the proliferation was inhibited to 49.9 ± 2.15% and 33.76 ± 2.7% after 48 and 72 h, respectively, with the IC50 value of 100.13 ± 2.1 ␮g ml−1 . Further, actinomycin D (1 ␮g ml−1 , positive control), a known anticancer drug, inhibited the proliferation of PBMC to 26.74 ± 2.17 after 48 h and DMSO (0.2%, negative control) did not inhibit the proliferation of PBMC, suggesting that Kocuran-mediated suppression of proliferation is not due to DMSO. The cell viability assessment by trypan blue exclusion method confirmed that the inhibitory effect on PHA-stimulated PBMC proliferation is not due to the toxicity of Kocuran (Supplementary Fig. S11). Further, Kocuran showed no significant effect on the proliferation of unstimulated PBMC (Supplementary Fig. S11). The inhibitory effect of Kocuran on PHAstimulated PBMC proliferation suggested that it can suppress the cytokine or mitogen-stimulated proliferation of PBMC. 3.7. Inhibition of complement mediated hemolysis Kocuran was demonstrated to inhibit both classical and alternative pathway mediated hemolytic reactions in a concentrationdependant manner (Supplementary Table S4). In the sensitized sheep erythrocytes the hemolysis is initiated by the classical complement pathway activation, while in case of rabbit erythrocytes the hemolysis is majorly initiated by the alternative complement pathway activation [30]. The CH50 and AH50 values representing the concentration required for 50% inhibition of classical and alternative pathway mediated hemolysis were 100.96 ± 1.75 and 98.60 ± 1.93 ␮g ml−1 , respectively. The complement mediated hemolysis is initiated by the formation of MAC that is covalently integrated on to the acceptor molecule or on cell surfaces through the ester or amide linkages formed with C3b fragment [46]. Further it is speculated that the observed inhibitory effect of Kocuran on the complement mediated hemolysis can be due to the hydrolysis of the thioester bonds in the C3 molecule or by the inhibition of complement activation cascade pathway [47]. 3.8. Binding ability of Kocuran to LPS The binding ability of Kocuran to LPS was determined by measuring the efficacy of Kocuran to inhibit the LPS-induced activation

Fig. 8. Binding ability of Kocuran to LPS. (a) The ability of Kocuran to bind to LPS was assessed using a quantitative chromogenic LAL assay. The LPS-induced activation of LAL enzyme was measured by incubating LPS (1 ␮g ml−1 ) in the presence of Kocuran at different concentrations (1, 10, 100 and 200 ␮g ml−1 ) and Polymyxin B (10 ␮g ml−1 , positive control) and the absorbance was measured at 410 nm. (b) Flow cytometric analysis of the effect of Kocuran on the binding of FITClabeled LPS to RAW264.7 macrophages. RAW macrophages were incubated with LPS-FITC (1 ␮g ml−1 ) in the absence (blue peak) or in the presence of Kocuran at 10 ␮g ml−1 (black peak), 100 ␮g ml−1 (red peak) and 200 ␮g ml−1 (thick green peak) and Polymyxin B, 10 ␮g ml−1 (light green peak). The darkened peak represents the background of untreated macrophages that was not treated with either LPS or Kocuran (dark peak). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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of LAL enzyme. Kocuran did not inhibit the LPS-induced LAL enzyme even at 200 ␮g ml−1 , suggesting that Kocuran is not interacting with LPS. However, Polymyxin B treatment showed significant inhibition of LPS-induced LAL enzyme (Fig. 8a). It was earlier reported that polymyxin B, a cationic cyclic peptide antibiotic, inhibits biological activities of LPS through high-affinity binding to lipid A moiety [48,49]. Further, when RAW macrophages were incubated with FITC-LPS (1 ␮g ml−1 ) in the presence of Kocuran, the binding of FITC-LPS to macrophages was not inhibited (Fig. 8b). The flow cytometric analysis revealed that there was no significant change in the FITC-LPS binding to RAW macrophages when the cells were incubated with Kocuran at different concentrations (10, 100 and 200 ␮g ml−1 ). The fluorescence of RAW macrophages treated with FITC-LPS and incubated with the presence of Kocuran was similar to the fluorescence of the cells incubated with the same amount of FITC-LPS without Kocuran. However, the pretreatment of RAW macrophages with Polymyxin B significantly inhibited the binding of FITC-LPS to RAW macrophages. 4. Conclusion Kocuran, an exopolysaccharide isolated from K. rosea strain BS1, has repeating monosaccharide residues of glucose, galactose, mannose and glucuronic acid. The in vitro models demonstrated the efficacy of Kocuran to suppress the release of macrophagederived ROS, NO, TNF-␣, IL-6 and C3 complement component from LPS-stimulated RAW 264.7 macrophages. Kocuran also inhibited the proliferation of PHA-stimulated human PBMC and complement mediated hemolysis suggesting its in vitro immunosuppressant activity. Further studies to understand the immunopharmacological role of Kocuran and the mechanism of action in both in vitro and in vivo systems are in progress. Acknowledgements

[9]

[10]

[11]

[12]

[13]

[14]

[15] [16]

[17]

[18]

[19]

[20]

[21]

[22] [23]

The authors acknowledge the financial assistance provided to PS in the form of Senior Research Fellowship by Council of Scientific and Industrial Research (CSIR), New Delhi, India.

[24] [25]

Appendix A. Supplementary data [26]

Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/ j.enzmictec.2013.10.007.

[27]

[28]

References [1] Genilloud O, Gonzalez I, Salazar O, Martin J, Tormo JR, Vicente F. Current approaches to exploit actinomycetes as a source of novel natural products. Journal of Industrial Microbiology and Biotechnology 2011;38: 375–89. [2] Lee HH, Lee JS, Cho JY, Kim YE, Hong EK. Study on immunostimulating activity of macrophage treated with purified polysaccharides from liquid culture and fruiting body of Lentinus edodes. Journal of Microbiology and Biotechnology 2009;19:566–72. [3] Sutherland IW. Structure-function relationships in microbial exopolysaccharides. Biotechnology Advances 1994;12:393–448. [4] Okutani K. Antitumor and immunostimulant activities of polysaccharides produced by a marine bacterium of the genus Vibrio. Bulletin of the Japanese Society of Scientific Fisheries 1984;50:1035–7. [5] Sun C, Wang JW, Fang L, Gao XD, Tan RX. Free radical scavenging and antioxidant activities of EPS2, an exopolysaccharide produced by a marine filamentous fungus Keissleriella rosea YS 4108. Life Sciences 2004;75:1063–73. [6] Brown DM, Donaldson K, Borm PJ, Schins RP, Dehnhardt M, Gilmour P, et al. Calcium and ROS-mediated activation of transcription factors and TNFalpha cytokine gene expression in macrophages exposed to ultrafine particles. American Journal of Physiology, Lung Cellular and Molecular Physiology 2004;286:L344–53. [7] Adams DO, Hamilton TA. The cell biology of macrophage activation. Annual Review of Immunology 1984;2:283–318. [8] Mai GT, Seow WK, McCormack JG, Thong YH. In vitro immunosuppressive and anti-phagocytic properties of the exopolysaccharide of mucoid strains of

[29]

[30] [31] [32] [33]

[34] [35]

[36]

[37] [38]

119

Pseudomonas aeruginosa. International Archives of Allergy and Applied Immunology 1990;92:105–12. Bleau C, Monges A, Rashidan K, Laverdure JP, Lacroix M, Van Calsteren MR, et al. Intermediate chains of exopolysaccharides from Lactobacillus rhamnosus RW-9595 M increase IL-10 production by macrophages. Journal of Applied Microbiology 2010;108:666–75. Nazina TN, Sokolova DS, Grigoryan AA, Xue Y-F, Belyaev SS, Ivanov MV. Production of oil-processing compounds by microorganisms from the Daqing oil field, China. Microbiology 2003;72:206–9. Dubey KV, Charde PN, Meshram SU, Shendre LP, Dubey VS, Juwarkar AA. Surface-active potential of biosurfactants produced in curd whey by Pseudomonas aeruginosa strain-PP2 and Kocuria turfanesis strain-J at extreme environmental conditions. Bioresource Technology 2012;126:368–74. Kumar CG, Joo HS, Choi JW, Koo YM, Chang CS. Purification and characterization of an extracellular polysaccharide from haloalkalophilic Bacillus sp. I-450. Enzyme and Microbial Technology 2004;34:673–81. Kumar CG, Joo HS, Kavali R, Choi JW, Chang CS. Characterization of an extracellular biopolymer flocculant from haloalkalophilic Bacillus isolate. World Journal of Microbiology and Biotechnology 2004;20:837–43. Kumar CG, Mamidyala SK, Sujitha P, Muluka H, Akkenapally S. Evaluation of critical nutritional parameters and their significance in the production of rhamnolipid biosurfactants from Pseudomonas aeruginosa BS-161R. Biotechnology Progress 2012;28:1507–16. Chaplin MF, Kennedy JF. Carbohydrate analysis: a practical approach. Washington, DC: IRL Press; 1986. p. 129–36. Dubois M, Gilles KA, Hamilton JK, Rebers PA, Smith F. Colorimetric method for determination of sugars and related substances. Analytical Chemistry 1956;28:350–6. Bradford MM. Rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 1976;72:248–54. Sweeley CC, Bentley R, Makita M, Wells WW. Gas liquid chromatography of trimethylsilyl derivatives of sugars and related substances. Journal of the American Chemical Society 1963;85:2497–507. Fontaine T, Fournet B, Karamanos Y. A new procedure for the reduction of uronic acid containing polysaccharides. Journal of Microbiological Methods 1994;20:149–57. Mort AJ, Bauer WD. Application of two new methods for cleavage of polysaccharides into specific oligosaccharide fragments. Journal of Biological Chemistry 1982;257:1870–5. Hakomori S. A rapid permethylation of glycolipid and polysaccharide catalyzed by methylsulfinyl carbanion in dimethyl sulfoxide. Journal of Biochemistry 1964;55:205–8. Angyal SJ, James K. Oxidation of carbohydrates with chromium trioxide in acetic acid. I. Glycosides. Australian Journal of Chemistry 1970;23:1209–21. Ara N, Hasan N. In vitro antioxidant activity of methanolic leaves and flowers extracts of Lippia alba. Research Journal of Medicine and Medical Sciences 2009;4:107–10. Liu F, Ooi VE, Chang ST. Free radical scavenging activities of mushroom polysaccharide extracts. Life Sciences 1997;60:763–71. Zhang Q, Yu P, Li Z, Zhang H, Xu Z, Li P. Antioxidant activities of sulfated polysaccharide fractions from Porphyra haitanesis. Journal of Applied Phycology 2003;15:305–10. Oyaizu M. Studies on product of browning reaction prepared from glucoseamine. Japanese Journal of Nutrition 1986;44:307–15. Wang H, Joseph JA. Quantifying cellular oxidative stress by dichlorofluorescein assay using microplate reader. Free Radical Biology and Medicine 1999;27:612–6. Zhang M, Tang H, Guo Z. Splenic stroma drives mature dendritic cells to differentiate into regulatory dendritic cells. Nature Immunology 2004;5: 1124–33. Mosmann T. Rapid colorimetric assay for cellular growth and survival; application to proliferation and cytotoxicity assays. Journal of Immunological Methods 1983;65:55–63. Platts-Mills TAE, Ishizaka K. Activation of the alternative pathway of human complement by rabbit cells. Journal of Immunology 1974;113:348–58. Yu L. Free radical scavenging properties of conjugated linoleic acids. Journal of Agricultural and Food Chemistry 2001;49:3452–6. Fridovich I. Biological effects of the superoxide radical. Archives of Biochemistry and Biophysics 1986;247:1–11. Mao H, Schnetz-Boutaud NC, Weisenseel JP, Marnett LJ, Stone MP. Duplex DNA catalyzes the chemical rearrangement of a malondialdehyde deoxyguanosine adduct. Proceedings of the National Academy of Sciences of the United States of America 1999;96:6615–20. Marnett LJ. Lipid peroxidation – DNA damage by malondialdehyde. Mutation Research 1999;424:83–95. Ma J, Chen T, Mandelin J, Ceponis A, Miller NE, Hukkanen M, et al. Regulation of macrophage activation. Cellular and Molecular Life Sciences 2003;60: 2334–46. Tripathi S, Maier KG, Bruch D, Kittur DS. Effect of 6-gingerol on proinflammatory cytokine production and costimulatory molecule expression in murine peritoneal macrophages. Journal of Surgical Research 2007;138:209–13. Alderton WK, Cooper CE, Knowles RG. Nitric oxide synthases: structure, function and inhibition. Biochemical Journal 2001;357:593–615. Goossens V, Grooten J, De Vos K, Fiers W. Direct evidence for tumor necrosis factor-induced mitochondrial reactive oxygen intermediates and their

120

[39]

[40] [41]

[42] [43]

C.G. Kumar, P. Sujitha / Enzyme and Microbial Technology 55 (2014) 113–120 involvement in cytotoxicity. Proceedings of the National Academy of Sciences of the United States of America 1995;92:8115–9. Scheurich P, Thoma B, Ucer U, Pfizenmaier K. Immunoregulatory activity of recombinant human tumor necrosis factor (TNF)-␣: induction of TNF receptors on human T cells and TNF-␣-mediated enhancement of T cell responses. Journal of Immunology 1987;138:1786–90. Carroll MC. The role of complement and complement receptors in induction and regulation of immunity. Annual Review of Immunology 1988;16:545–68. Sahu A, Kozel TR, Pangburn MK. Specificity of the thioester-containing reactive site of human C3 and its significance to complement activation. Biochemistry 1994;302:429–36. Frank MM, Fries LF. The role of complement in inflammation and phagocytosis. Immunology Today 1991;12:322–9. Ranges GE, Zlotnick A, Esperik T, Dinarello CA, Cerami A, Pallandino Jr MA. Tumor necrosis factor alpha/cachectin is a growth factor for thymocytes: synergistic interactions with other cytokines. Journal of Experimental Medicine 1988;167:1472–8.

[44] Charles Jr AJ, Paul T, Hunt S, Walport M. Immunobiology. New York: Current Biology Ltd.; 1997. [45] Janeway CA, Travers P, Hunt S, Walport M. Immunobiology: the immune system in health and disease. New York: Garland Publishing Inc; 1997. [46] Muller-Eberhard HJ, Dalmasso AP, Calcott MA. The reaction mechanism of ␤ 1c-globulin (C3) in immune hemolysis. Journal of Experimental Medicine 1966;123:33–54. [47] Pangburn MC, Schreiber RD, Muller-Eberhard HJ. Formation of the initial C3 convertase of the alternative pathway: acquisition of C3b-like activities by spontaneous hydrolysis of the putative thioester in native C3. Journal of Experimental Medicine 1977;146:257–70. [48] Coyne CP, Fenwick BW. Inhibition of lipopolysaccharide-induced macrophage tumor necrosis factor-␣ synthesis by polymyxin B sulfate. American Journal of Veterinary Research 1993;54:305–14. [49] Morrison DC, Jacobs DM. Binding of polymyxin B to the lipid A portion of bacterial lipopolysaccharides. Immunochemistry 1976;13: 813–8.