Effect of bursal anti-steroidogenic peptide and immunoglobulin G on neonatal chicken B-lymphocyte proliferation

Effect of bursal anti-steroidogenic peptide and immunoglobulin G on neonatal chicken B-lymphocyte proliferation

Comparative Biochemistry and Physiology Part C 134 (2003) 291–302 Effect of bursal anti-steroidogenic peptide and immunoglobulin G on neonatal chicke...

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Comparative Biochemistry and Physiology Part C 134 (2003) 291–302

Effect of bursal anti-steroidogenic peptide and immunoglobulin G on neonatal chicken B-lymphocyte proliferation R.W. Moorea,*, D.Y. Caldwella, L.R. Berghmana, D.J. Caldwella, A.P. McElroya,1, J.A. Byrdb, B.M. Hargisa,2 a b

Departments of Poultry Science and Veterinary Pathobiology, Texas A&M University, College Station, TX 77843, USA United States Department of Agriculture, Agricultural Research Service, Southern Plains Agricultural Research Center, College Station, TX 77845, USA Received 31 May 2002; received in revised form 22 September 2002; accepted 23 September 2002

Abstract In attempts to identify antibodies for Bursal Anti-Steroidogenic Peptide (BASP), rabbit serum was observed to reduce phorbol ester-stimulated chicken B-lymphocyte proliferation comparable to BASP. These experiments investigated the effects of IgG on B-lymphocyte proliferation. In Experiment 1, 3% rabbit serum decreased B-lymphocyte proliferation. In Experiment 2, 2 mgyml of intact rabbit IgG or 0.65 mgyml of IgG papain digest products, Fab and Fc, decreased Blymphocyte proliferation. The combination of BASP and either Fab or Fc was observed to have at least an additive antiproliferative effect. In Experiment 3, 0.01 mgyml of either rabbit or chicken IgG, or 1.0 mgyml of rabbit or 0.01 mgy ml of chicken Fab, Fc, and the pepsin digestion product F(ab9)2 was observed to have an anti-proliferative effect. No combined effects of BASP and IgG or IgG digest products were observed for this experiment. In Experiment 4, 12 mgy ml of chicken egg yolk IgG or 1.2 mgyml Fab was found to suppress B-lymphocyte proliferation. Additionally, an additive effect of 12 mgyml of IgG with BASP was again observed. The present studies suggest that IgG and its digestion products reduce phorbol-stimulated B-lymphocyte proliferation in vitro and combined treatment with IgG and BASP may have at least an additive anti-proliferative effect on B-lymphocyte proliferation. 䊚 2002 Elsevier Science Inc. All rights reserved. Keywords: B-lymphocyte; Bursal anti-steroidogenic peptide; Immunoglobulin G; Proliferation

1. Introduction Our laboratory has previously identified and partially characterized a bioactive protein with an apparent molecular weight of 30 kDa from the *Corresponding author. Department of Poultry Science, Texas A&M University, College Station, TX 77845, USA. Tel.: q1-979-845-4818; fax: q1-979-845-1931. E-mail address: [email protected] (R.W. Moore). 1 Present Address: Department of Animal and Poultry Sciences, Virginia Tech, Blacksburg, VA 24061, USA. 2 Present Address: Department of Poultry Science, University of Arkansas, Fayetteville, AR 72701, USA.

chicken bursa of Fabricius which we have named Bursal Anti-Steroidogenic Peptide (BASP; Byrd et al., 1993). In addition to marked anti-steroidogenic properties (Byrd et al., 1993, 1994, 1995; Dean et al., 1995a,b), BASP also has marked anti-proliferative effects on mitogen-stimulated neonatal bursal-derived lymphocytes (Caldwell et al., 1999). This rapid bioassay, which measures tritiated thymidine (w3HxTdR) uptake, has been used for measuring BASP bioactivity during purification. In an attempt to develop polyclonal antibodies for further characterization of BASP, several rab-

1532-0456/03/$ - see front matter 䊚 2002 Elsevier Science Inc. All rights reserved. PII: S 1 5 3 2 - 0 4 5 6 Ž 0 2 . 0 0 2 0 8 - 9

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bits were hyperimmunized against BASP. However, preliminary experiments using serum from either normal or BASP-immunized rabbits reduced mitogen-stimulated neonatal chick B-lymphocyte proliferation in vitro. This led to the hypothesis that heat-treated rabbit serum may contain a substance which has a marked anti-proliferative effect on mitogen-stimulated neonatal B-lymphocytes. Similar anti-proliferative activity of non-specific heat-treated rabbit serum control samples during further preliminary attempts to test for neutralization of BASP utilizing purified rabbit antibodies led us to conclude that this substance may actually be immunoglobulin G (IgG). A possible role of IgG in the control of immature chicken B-lymphocyte differentiation and selection was first hypothesized by Glick and Olah (1993). In this hypothesis, B-lymphocytes with correctly rearranged surface IgM must come in contact with IgG which is presented on the surface of the bursal secretory dendritic cell, an action that prompts the release of an unknown cytokine which modulates differentiation of the immature B-lymphocytes in the bursal milieu. Hypothetically, such differentiation may slow proliferative responses of affected cells (Freshney, 1983). Additionally, a feed-back mechanism of IgG and IgG fragments on lymphocyte proliferation has been proposed in human lymphocytes (Klaesson et al., 1996; Zhuang and Mazer, 2001). The following investigation was initiated to test the hypothesis that IgG, or constituent components of IgG, could directly influence in vitro mitogen-stimulated proliferation of neonatal chick B-lymphocytes. 2. Materials and methods 2.1. Purification of bursal anti-steroidogenic peptide Extraction of bioactive bursal extract from bursal tissue. A number of protocols for purification of bioactive BASP have been evaluated by our laboratory, each using a combination of the published bioassay techniques described below (Byrd et al., 1993; Caldwell et al., 1999). The purification procedure described here has consistently yielded highly bioactive material. Briefly, bursae of Fabricius were removed from 7-week-old chickens, immediately frozen on dry ice, and stored at y76 8C prior to extraction. Tissue was homogenized in two parts 15% trifluoroacetic acid (Sig-

ma) using a commercial blender for 5 min. The tissue homogenate was centrifuged for 10 min at 37 000=g and the supernatant was carefully removed, avoiding contamination with surface lipid. The supernatant was then loaded onto 6 separate 10-g standard size solid phase extraction cartridges (Varian Associates). Cartridges were eluted with 15 ml of each of the following: 5% acetonitrile (ACN; HPLC Grade, EM Science)-5 mM TFA, 30% ACN-5 mM TFA (vyv), 60% ACN-5 mM TFA and 80% ACN-5 mM TFA. The elution containing the anti-steroidogenic and anti-proliferative activity has consistently been found in the 30% ACN-5 mM TFA fraction. This elution was filtered (0.2 mm) and tested for bioactivity, utilizing the cell cultures systems described below. The bioactive elutions were lyophilized and stored at y20 8C until further purification. For simplicity, we will refer to the resulting material at this stage of purification as crude BASP (cBASP). Preparative C-18 reversed phase HPLC (rpHPLC). A subsample of cBASP was diluted 15fold with 5 mM TFA for fractionation by rpHPLC. The 5-mM TFA-diluted sample was injected onto a 5-mM TFA equilibrated preparative C-18 column (Waters Chromatography) with separation of bursal extracts occurring at a flow rate of 7 mlymin from a generated gradient of 0% to 80% ACN. Sixty 1-min fractions were collected into 12=75mm polypropylene test tubes and dried by vacuum centrifugation. Even numbered fractions were resolubilized in appropriate cell culture medium at the time of bioassay. Suppression of mitogenstimulated bursal lymphocyte DNA synthesis was used for determining bioactive fractions during all purification procedures as described below. Following bioassay, fractions with observable bioactivity were pooled for further purification. For simplicity, we will refer to the resulting material at this stage of purification as partially purified BASP (ppBASP). Cation exchange chromatography. Twenty-five grams of cation exchange medium (Accell Plus, Waters Chromatography) was used in a 1.5=20cm glass column and washed with 10 volumes of ddH2O. Pooled bioactive fractions of ppBASP were fractionated by collecting the first 35 ml elution immediately following the sequential addition of 235 ml of either ddH2O, 0.375, 0.75 or 1.5 M ammonium bicarbonate. Following bioassay, bursal extract bioactivity has been consistently

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identified in the 0.75-M ammonium bicarbonate fraction. This fraction was concentrated and buffer exchanged with ddH2O using a 10-kDa exclusion membrane (Centriprep Concentrator, Amicon). For simplicity, we will refer to the resulting material at this final stage of purification as highly purified BASP (hpBASP). This bioactive material was used in the B-lymphocyte culture experiments. SDS PAGE for analysis of bursal anti-steroidogenic peptide. Biologically active hpBASP was applied to SDS PAGE using a 12.5% homogeneous gel (Pharmacia Biotech) with appropriate SDS buffer strips using a LKB Phast System (Pharmacia Biotech). 2.2. Bioassay 2.2.1. Cell culture media for B-lymphocyte cultures The basic cell culture medium for all lymphocyte cultures consisted of RPMI 1640 with Lglutamine (Sigma) at a final pH of 7.2. The complete medium also contained 2 gyl sodium bicarbonate, 0.1% bovine serum albumin (Sigma), 100 unitsyml penicillin and 100 mgyml streptomycin (Gibco BRL), and 50 mgyml gentamicin (Schering-Plough Animal Health). 2.2.2. Isolation and culture of neonatal bursal lymphocytes Approximately 20 1-day-old single-comb white Leghorn (SCWL; Hy-Line W36䉸) chicks were obtained from a local commercial hatchery (HyLine International, Bryan, TX). Immediately following CO2 asphyxiation, the feathers were moistened with 70% ethanol, and chicks were placed under a laminar flow hood for dissection and excision of cloacal bursae. The bursa of Fabricius of each chick was excised at the proctodeal junction and immediately placed into a sterile 50-ml conical centrifuge tube containing 25 ml of RPMI 1640. All subsequent steps in the bursal lymphocyte isolation procedure were performed on wet ice at approximately 4 8C. After removal from carcasses, bursae were transferred to a sterile Petri dish containing approximately 5 ml of RPMI 1640. The bursae of Fabricius were gently teased apart with 18 gauge needles until a suspension of small tissue fragments was obtained. Tissue fragments from all birds were pooled and transferred to a sterile 50-ml conical tube where they were gently triturated with four sterile 5-cc syringes with progressively smaller inlets (approx-

293

imately 8, 6, 4 or 2-mm inlet sizes produced by trimming the luer portion of the syringe with a scalpel). Beginning with the syringe with the largest inlet, tissue was titurated until the resulting cell suspension was noticeably opaque, at which time, tissue fragments received a total of about 30 titurations per syringe. The resulting cell suspension was filtered to remove clumps of cells or tissue fragments by transfer to a sterile 50-ml conical tube covered with nylon mesh of 60 mm pore size (Nytex, Tetko Inc.). The filtered cell suspension was then pelleted by centrifugation for 10 min at 350=g at 4 8C. Cells were washed by resuspension in fresh RPMI 1640 (4 8C) and repelleted by centrifugation. Cells were washed a total of three times prior to assessment of number and viability. Immediately following the final washing step, a 100-ml aliquot of the cell suspension was subjected to the live-dead staining procedure of Freshney (1983), and the final concentration of cells in suspension was adjusted to 4=106 viable cellsyml with fresh RPMI 1640. Viability of isolated cells was )90% in all experiments. 2.2.3. Analysis of DNA synthesis In all experiments, cell cultures were carried out in 96-well polystyrene tissue culture plates (Falcon). Treatments, mitogen, cells, and w3HxTdR (ICN Pharmaceuticals; 1:199 dilution of w3HxTdR, specific activity 6.7 Ciymmol and 1 mCiyml, with cell culture medium) was added to each well in 50-ml volumes so that the final volume in all wells in each experiment was 250 ml. Six replicate wells were used for each treatment. Cultures were incubated at 37 8C, with saturated humidity and 5% CO2, for 24 h. Isolated B-lymphocytes were exposed to mitogen (phorbol 12,13-dibutyrate; Sigma) andyor treatments for 16 h prior to the addition of w3HxTdR. Following these initial incubation periods, cells were pulsed with w3HxTdR for the final 8 h of the entire culture period. At termination, cells were harvested onto filter mats (Skatron Instruments) using a semi-automatic 12 well cell harvester (Skatron Instruments). Filter mats were dried, the filter discs corresponding to each well were placed into 7-ml polypropylene scintillation vials, and 5 ml of an aqueous-based scintillation counting cocktail (Ultima Gold, Packard Instrument) was added. Radioactivity was counted for 2 min in a standard liquid scintillation counter (Beckman Instruments). Counts were auto-

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matically quench corrected and data were reported as disintegrations per minute (dpm). In Experiment 2, an additional wellytreatment was subjected to the previously mentioned live-dead staining procedure at the time of cell harvesting. 2.3. Experiments 2.3.1. Effect of Rabbit Serum (Experiment 1) Serum was obtained from 3 rabbits (2 hyperimmunized against crude bursal extracts and 1 non-immunized animal) for evaluation in B-lymphocyte cell culture. The serum samples were heated at 55–60 8C for 15 min to destroy complement activity and were maintained at y20 8C prior to use. Samples were diluted in culture medium and added in 50 ml volumes to obtain final concentrations of 0, 0.3, 1 and 3% serumywell. 2.3.2. Effect of purified rabbit IgG, Fc and Fab (Experiment 2) Commercially purified rabbit IgG (rIgG; Sigma) was enzymatically digested as follows: rabbit IgG was dissolved in 0.1 M Na acetate (5 mg rIgGy1 ml Na acetate; Sigma) and 0.5 mlyml of 1 M cysteine (Sigma) and 0.5 mlyml of 20 mM EDTA (Sigma) were added. The solution was mixed thoroughly and 50 mlyml of papain (1 mg papainy ml of 0.1 M Na acetate; Sigma) was added. The solution was mixed and incubated at 37 8C in a shaking H2O bath for 12 h. One hundred mlyml of 75 mM iodoacetamide (Sigma) was added to the solution to stop the enzymatic reaction, and the solution was incubated at room temperature for an additional 30 min. Antibody Fc fragments were separated from the Fab fragments using a protein A column (Sigma; 2 ml of protein A beads in a disposable polypropylene column). Fragment purity was analyzed with SDS-PAGE. Fragment solutions were dialyzed against RPMI 1640 medium using a 10 kDa ultrafiltration membrane (Slide-A-Lyzer, Pierce). Whole rIgG was diluted in RPMI 1640 medium to obtain a final estimated concentration in the wells of 2 mgyml (0.5 mgy well). Rabbit Fc (rFc) and Fab (rFab) fragments were diluted to obtain a similar molar concentration as the rIgG (0.65 mgyml). All dilutions were made assuming no loss of products in the digestion, dialysis and fragment purification procedures. Therefore, actual concentrations were likely somewhat less than these estimated concentrations.

2.3.3. SDS-Page Digested antibodies were separated by both reducing and non-reducing SDS-PAGE, respectively, on an 8=8 cm 10% Bis-Tris NuPAGE娃 gel (Novex, San Diego, CA) according to the manufacturer’s instructions. Briefly, samples were diluted with NOVEX NuPAGE娃 4x denaturing sample buffer (pH 8.5) with estimated total protein concentrations ranging between 1 and 5 mg per well. The samples for separation under reducing conditions were supplemented with NuPAGE reducing agent (dithiothreitol (DTT), 50 mM final concentration) just prior to heating and loading the samples. All samples were heated for 10 min at 70 8C, vortexed and loaded into the sample wells. Both buffer compartments of the apparatus were filled with the 50 mM Tris–MOPS (3-(N-morpholino) propane sulfonic acid) (pH 7.7) running buffer containing 0.1% (wyv) SDS and 0.03% EDTA. Electrophoresis was performed under constant voltage at 200 V for 35 min. Upon separation, the gels were fixed in 50% methanol, 10% acetic acid in MilliQ water for 10 min and stained with the NOVEX Colloidal Blue Kit according to the instructions of the manufacturer. 2.3.4. Effect of commercially prepared rabbit IgG, Fc, F(ab9)2 and Fab (Experiment 3) The following commercially purified IgG products were obtained from Rockland Immunochemicals: whole rIgG, rFc, rFab, rabbit F(ab9)2, (rF(ab9)2) which is obtained from pepsin digestion of rIgG, whole chicken IgG (cIgG), chicken Fc fragment (cFc), chicken Fab fragment (cFab) and chicken F(ab9)2 fragment (cF(ab9)2 ). Each of these products were dialyzed against RPMI 1640 medium using a 10-kDa membrane (Pierce) and diluted to a putative final concentration of 0.01, 0.1 or 1 mgyml (0.0025, 0.025 and 0.25 mgywell) assuming no loss of products in the dialysis procedures. BSA (Sigma) was also evaluated at either 0, 0.01, 0.1 or 1 mgyml final concentrationsywell to determine the effect of added non-immunoglobulin protein on the parameters measured in these experiments. 2.3.5. Effect of chicken IgG, Fab, Fc. (Experiment 4) Chicken IgG was purified from egg yolks with an ammonium sulfate ((NH4)2SO4 ) precipitation procedure which has been previously described (Jensenius and Koch, 1997). Briefly, 12 eggs were

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frozen overnight at y20 8C. Egg yolks were collected by removing shell and frozen albumen. Egg yolks were thawed to RT and diluted to 1 l with ddH2O (1:10 wt.yvol.). The yolk suspension was stirred for 2 h at RT and then the pH was adjusted to 7.0 with NaOH. The mixture was placed in six 250-ml Nalgene centrifuge containers and frozen at y20 8C overnight. Solutions were thawed and centrifuged at 3000=g at 4 8C for 20 min. The supernatant was collected in 50-ml conical tubes, cooled for 10 min at y80 8C to improve precipitation, and re-centrifuged at 2000=g at 4 8C for 15 min. Supernatant was again collected into a beaker, pH was adjusted to 7.6 with NaOH, and the supernatant was filtered across a 0.8 mm filter. Non-immunoglobulin proteins were precipitated by adding 90 mg of (NH4)2SO4 per ml of supernatant, stirring for 30 min at RT, and centrifuging in 50-ml conical tubes at 2500=g for 30 min at 4 8C. To precipitate the cIgG, another 90 mg of (NH4)2SO4 per ml of supernatant was added slowly over a 5-min period, and the solution was stirred for an additional 1 h at RT. The resulting mixture was placed in 50-ml conical tubes and incubated for an additional hour at 4 8C. The conical tubes containing the solution were centrifuged at 2500=g for 30 min at 4 8C, the supernatant was discarded, and the precipitate was dissolved in 80 ml of 0.14 M NaCl, 10 mM Tris– HCl, pH 7.4 (TBS) and then placed into two 50ml conical tubes. The cIgG was re-precipitated by adding 180 mg of (NH4)2SO4 per ml of solution, incubating the solution at 4 8C for 2 h, and centrifuging the tubes at 2500=g for 30 min at 4 8C. The precipitate was collected and dissolved in a total of 25 ml of TBS. Twenty-five milliliters of 3.6 M (NH4)2SO4 was added to the conical tube, and the solution was incubated for 2 h at 4 8C. The solution was centrifuged at 2500=g for 30 min at 4 8C and the supernatant was discarded. The precipitate was then dissolved in 25 ml of 10 mM Na2HPO4, 150 mM NaCl, pH 7.2 (PBS). The cIgG solution was dialyzed against PBS and then lyophilized and stored at y20 8C. Approximately 400 mg of product was obtained. This product was estimated to contain 0.63 g of cIgG per gram of product using a commercial protein assay (BioRad) and a rIgG standard. A portion of the cIgG was enzymatically digested with papain to obtain the Fab IgG fragment using the methods of Akita and Nakai (1993). Briefly, papain digestion was performed as follows:

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150 mg of the cIgG product was diluted in 9.5 ml of 100 ml Na2HPO4, pH 7.0 with 3 mM EDTA and 10 mM cysteine (PB) in a 15-ml conical tube. Ninety-five microliters of a stock solution of papain (10.25 mg papainyml of 100 mM PB; Sigma) and the solution was incubated at 37 8C for 17 h. The reaction was terminated by adding 136 ml of a stock solution of iodoacetamide (0.4 g C2H4INOyml ddH2O) and incubating the solution for 30 min at 37 8C. The digest was stored at 4 8C until purification of cFab and cFc fragments. The IgG fragment mixture was purified using a 2=4 cm DEAE-Sephacel (Amersham Pharmacia Biotech) column. The Fab fragment was eluted with 100 mM Tris HCl, pH 8.0. The Fc fragment was collected after the Fab fragment using 100 mM Tris HCl containing 200 mM NaCl, pH 8.0. Elutions were collected in 1-ml fractions, and fractions containing fragments were detected at 280 nm. Fractions of like fragments were pooled and dialyzed against 5 changes of RPMI 1640, 500 ml each, using 10-kDa dialysis cassettes. Dialyzed fragments were stored at y20 8C prior to use in experiments. Intact cIgG, Fab and Fc were each diluted to 6 mgyml for use in the Blymphocyte culture based on estimated protein content determined using the commercial protein assay with rIgG standards, resulting in a final concentration in the wells of 1.2 mgyml (0.3 mgy well). Additionally, cIgG was used at a final concentration in the wells of 12 mgyml (3 mgy well) in this experiment. 2.3.6. Statistical analysis All data within experiments were analyzed using the General Linear Model procedure for analysis of variance (SAS Institute, 1996). Statistically different means (P-0.05) were further separated using Duncan’s Multiple Range Test (SAS Institute, 1996). In Experiment 3, data from rIgGy rIgG-fragment and cIgGycIgG-fragment evaluations were independently analyzed. 3. Results Bioactivity of the hpBASP was confirmed utilizing the B-lymphocyte cell culture prior to further experimentation (Fig. 1). Purity of the hpBASP was determined utilizing SDS PAGE (Fig. 2). 3.1. Experiment 1 Experiment 1 was initiated to examine the effect of heat-inactivated rabbit serum on the prolifera-

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tion of neonatal B-lymphocytes. Serum from a non-immunized rabbit (serum 噛1) and serum from two rabbits hyperimmunized to crude bursal extracts (serum 噛2 and 噛3) were utilized at either 0, 0.3, 1 or 3% final concentrations in the B-cell culture (Table 1). Serum from the non-immunized rabbit (serum 噛1) caused a small but significant increase in the proliferative response of the mitogen-stimulated B-lymphocytes at 0.3 and 1%, but markedly and significantly decreased the proliferation of cells at a final concentration of 3% (a greater than 16-fold reduction in DPM). Additionally, the serum from hyperimmunized animals caused a greater reduction in cell proliferation than that of the non-immunized animal. As little as 0.3% final concentration of serum from the hyperimmunized animals significantly decreased the proliferative response of the B-lymphocytes in this study. 3.2. Experiment 2 Experiment 2 was performed to examine the effect of commercially purified rIgG or purified enzymatic digestion products of rIgG on B-lymphocyte proliferation. Numbers of viable cells were found to be similar for all treatment groups at the time of cell harvesting. Analyses of enzymatic digestion of the commercially purified rIgG are shown in Fig. 3. In this experiment, either whole rIgG (2 mgyml estimated final concentration) or the papain rIgG digestion products of rFc and rFab (each used at the estimated concentration of 0.65

Fig. 2. SDS-PAGE gel (silver stained) bioactive, highly purified Bursal Anti-Steroidogenic Peptide (hpBASP). Lane A: Molecular mass markers; Lane B: 5 bursal equivalents (Beq) of hpBASP; Lane C: 1 Beq of hpBASP.

mgyml), were evaluated with the B-lymphocyte cell culture (Table 2). In this experiment, a 2 mgy ml concentration of commercially purified rIgG significantly decreased the proliferation of B-lymphocytes as measure by w3HxTdR uptake. Additionally, equal molar concentrations of each of the papain IgG digestion products, rFab or rFc, had a

Fig. 1. Effect of partially purified and highly purified Bursal Anti-Steroidogenic Peptide (ppBASP and hpBASP respectively; 12.5 bursal equivalents (Beq)yml) on Phorbol 12,13 Dibutyrate- (PDB; 25 ngyml) stimulated neonatal B-lymphocyte proliferation as measured by tritiated thymidine uptake. Each bar represents the mean Disintegrations per Min (DPM) of each respective treatment "S.E.M. (ns6 wells per treatment). Means with no common superscript differ significantly (P-0.05).

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Table 1 Effect of heat inactivated rabbit serum on proliferation of neonatal B-lymphocytes as measured by tritiated thymidine uptake1 (Experiment 1) Concentration of heat inactivated serum2 Treatment

0%

PDB qserum 噛1 PDB3qserum 噛25 PDB3qserum 噛35 3

4

0.3% c

26 747"450 26 747"450c 26 747"450c

1.0% a

30 118"276 23 293"622d 23 700"436d

3.0% b

28 293"552 3063"324e 3506"214e

1654"114f 200"26g 292"62g

1

Each number represents the mean disintegrations per minute of each respective treatment"SEM (ns6 wells per treatment). Means with no common superscript differ significantly (P-0.05). 2 Percent of heat inactivated serum added to neonatal B-lymphocyte cell culture. 3 Phorbol 12,13 dibutyrate (25 ngyml). 4 Serum obtained from a non-immunized rabbit. 5 Serum obtained from rabbits hyperimmunized to crude bursal extracts.

greater inhibitory effect on proliferation than did the intact IgG molecule. Bursal Anti-Steroidogenic Peptide was found to have a greater anti-proliferative effect than the tested concentrations of whole rIgG alone; however, rFab and rFc were observed to have a greater anti-proliferative effect than the concentration of BASP used in this study. Additionally, BASP in combination with either whole rIgG or rIgG fragments were found to have an at least an additive anti-proliferative effect.

3.3. Experiment 3 Experiment 3 was performed to further examine the effects of IgG on B-lymphocyte proliferation. In this experiment, commercially-purified IgG and both papain and pepsin digestion products of rabbit and chicken IgG were evaluated. The effect of each IgG product on mitogen-stimulated lymphocyte proliferation was examined at either 0, 0.01, 0.1 or 1 mgyml final concentration. Additionally, BSA was evaluated at either 0, 0.01, 0.1 or 1 mgy

Fig. 3. SDS PAGE gel of commercially-purified rabbit Immunoglobulin G (rIgG) and of rIgG papain digestion products Fab and Fc. Lanes C–F contain products which have been reduced with b-mercaptoethanol. Lanes H–L contain non-reduced products. Each lane contains approximately 35 mg of the following products: Lanes C and H contain papain digested products; Lanes D, I and L contain Fc fragment obtained from protein A purification; Lanes E and J contain whole rIgG; Lanes F and K contain Fab fragment obtained from protein A purification; Lanes A, B and G contain gel markers.

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Table 2 Effect of whole or digested rabbit Immunoglobulin G on proliferation of neonatal B-lymphocytes as measured by tritiated thymidine uptake1 (Experiment 2) Treatment

Control

rIgG2 (2 mgyml)

rFab3 (0.65 mgyml)

rFc4 (0.65 mgyml)

PDB5 PDB5qBASP6

161 137"1590a 69 165"1380c

87 975"3872b 3893"1374f

8873"2698d 212"12f

13 461"1994d 191"10f

1

Each number represents the mean disintegrations per minute of each respective treatment"S.E.M. (ns6 wells per treatment). Means with no common superscript differ significantly (P-0.05). 2 Rabbit immunoglobulin G. 3 Rabbit immunoglobulin G Fab fragment. 4 Rabbit immunoglobulin G Fc fragment. 5 Phorbol 12,13 dibutyrate (25 ngyml). 6 Highly purified Bursal Anti-Steroidogenic Peptide (10 Beqyml).

ml final concentrations to determine the effect of adding protein to the cell culture. The results of Experiment 3 are shown in Tables 3 and 4. Addition of the protein control, BSA, caused a small but significant depression of lymphocyte proliferation at either 0.1 or 1.0 mgyml (Tables 3 and 4). Rabbit IgG was found to have a significant anti-proliferative effect on B-lymphocytes at every concentration tested, as compared to either control or BSA treatment groups (Table 4). While all three enzyme digestion products of rIgG had a significant effect on the proliferation of B-lymphocytes at 0.1 or 1 mgyml as compared to control levels, the rIgG digestion fragments only caused suppression of proliferation greater than the BSA treatment

at the highest level (1.0 mgyml) tested (Table 3). No difference in magnitude of proliferation suppression induced by either of the rIgG digestion products was noted. Although BASP caused marked attenuation of lymphocyte proliferation in this experiment, no additive effect of BASP and rIgGyrIgG-fragment (Table 3) or cIgGycIgG-fragment (Table 4) combination were observed at the concentrations evaluated in this experiment. Chicken IgG and cIgG fragments also caused significant depression of lymphocyte proliferation as compared either to controls or BSA (Table 4). Interestingly, cF(ab9)2 caused the greatest magnitude of suppression of proliferation, with further reductions as compared to those induced by intact cIgG,

Table 3 Effect of commercially-prepared rabbit Immunoglobulin G products on proliferation of neonatal B-lymphocytes as measured by tritiated thymidine1 uptake (Experiment 3) Treatment

Control

rIgG2

PDB7 PDB7qBASP8

82 754"1448a 8397"630g

69 348"3100d 8754"194g

PDB7 PDB7qBASP8

82 754"1448a 8397"630g

60 915"3014e 6354"238g

PDB7 PDB7qBASP8

82 754"1448a 8397"630g

53 105"2762f 8602"264g

rFab3

rF(ab9)24

rFc5

Concentration of treatment (0.01 mgyml) 75 875"2010bc 76 211"2932bc 77 064"2932bc 7478"90g 6784"122g 7480"138g (0.1 mgyml) 73 901"1994cd 72 077"2884cd 72 511" 2182cd 6543"202g 6589"186g 7177"100g (1.0 mgyml) 57 508"1938ef 55 753"1142ef 54 254"528f 5041"220g 5110"80g 5034"152g

BSA6

79 888"1682ab 7553"166g 75 064"1820bcd 7595"152g 73 380"2898cd 8983"176g

1 Each number represents the mean disintegrations per minute of each respective treatment"S.E.M. (ns6 wells per treatment). Means with no common superscript differ significantly (P-0.05). 2 Rabbit immunoglobulin G. 3 Rabbit immunoglobulin G Fab fragment. 4 Rabbit immunoglobulin G F(ab9)2 fragment. 5 Rabbit immunoglobulin G Fc fragment. 6 Bovine serum albumin. 7 Phorbol 12,13 dibutyrate (25 ngyml). 8 Highly purified Bursal Anti-Steroidogenic Peptide (10 Beqyml).

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Table 4 Effect of commercially-prepared chicken Immunoglobulin G products on proliferation of neonatal B-lymphocytes as measured by tritiated thymidine uptake1 (Experiment 3) Treatment

CIgG2

Control

PDB7 PDB7qBASP8

82 754"1448a 8397"630k

72 072"2943cdef 6386"200k

PDB7 PDB7qBASP8

82 754"1448a 8397"630k

72 202"2886cdef 6767"172k

PDB7 PDB7qBASP8

82 754"1448a 8397"630k

63 065"6366ghi 5045"14k

cFab3

CF(ab9)24

cFc5

Concentration of immunoglobulin treatment (0.01 mgyml) 70 540"1116def 66 392"1882fgh 72 812"1731cde 8167"268k 10 025"262k 6777"101k (0.1 mgyml) 69 010"1186efg 61 386"2222hi 72 630"3398cde k k 7128"66 7896"126 6700"116k (1 mgyml) 58 918"1930i 33 850"580j 58 419"2538i 5427"96k 5941"110k 4791"92k

BSA6

79 888"1682ab 7553"166k 75 064"1820bc 7595"152k 73 380"2898cde 8983"176k

1

Each number represents the mean disintegrations per minute of each respective treatment"S.E.M. (ns6 wells per treatment). Means with no common superscript differ significantly (P-0.05). 2 Chicken immunoglobulin G. 3 Chicken immunoglobulin G Fab fragment. 4 Chicken immunoglobulin G F(ab9)2 fragment. 5 Chicken immunoglobulin G Fc fragment. 6 Bovine serum albumin. 7 Phorbol 12,13 dibutyrate (25 ngyml). 8 Highly purified Bursal Anti-Steroidogenic Peptide (10 Beqyml). Table 5 Effect of whole or digested chicken immunoglobulin G on proliferation of neonatal B-lymphocytes as measured by tritiated thymidine uptake1 (Experiment 4) Treatment

Control

PDB5 PDB5qBASP6

26 962"1325a 7673"332d

PDB5 PDB5qBASP6

26 962"1325a 7673"332d

cIgG2

cFab3

cFc4

Concentration of immunoglobulin treatment (1.2 mgyml) 24 721"1736a 18 543"957b 24 590"1471a 8180t"823d 3032"407e 12 740"676c (12 mgyml) 5872"561d ND7 ND7 2742"234e ND7 ND7

1 Each number represents the mean disintegrations per minute of each respective treatment"S.E.M. (ns6 wells per treatment). Means with no common superscript differ significantly (P-0.05). 2 Chicken immunoglobulin G purified from egg yolk. 3 Papain digested chicken immunoglobulin G Fab fragment purified from egg yolk. 4 Papain digested chicken immunoglobulin G Fc fragment purified from egg yolk. 5 Phorbol 12,13 dibutyrate (25 ngyml). 6 Highly purified Bursal Anti-Steroidogenic Peptide (10 Beqyml). 7 Not Determined.

at each concentration tested. Exposure of the lymphocytes to cFab also caused greater suppression of mitogen-induced lymphocyte proliferation than intact IgG at the 2 higher concentrations evaluated, while the magnitude of cFc-induced proliferation suppression was similar to the suppression caused by intact cIgG at each concentration (Table 4). 3.4. Experiment 4 Experiment 4 was performed to further examine the effects of selected concentrations of cIgG on

B-lymphocyte proliferation. In this experiment, cIgG was purified from egg yolks and papain digestion products were obtained. Each cIgG product was examined at 1.2 mgyml final concentration. Additionally, whole cIgG was evaluated at a final concentration of 12 mgyml. The results of Experiment 4 are shown in Table 5. Only the highest concentration of cIgG (12 mgyml) or cFab (1.2 mgyml) evaluated were found to significantly suppress B-lymphocyte proliferation as compared to controls in this experiment. Interestingly, cFab

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was again apparently more efficacious in suppressing proliferation of lymphocytes than either intact cIgG or cFc in this experiment. The addition of purified BASP consistently decreased the proliferation of lymphocytes as compared to the stimulated control. An apparent additive effect of combination 12 mgyml intact cIgG with BASP was observed, with significantly greater suppression of proliferation caused by this combination than by either cIgG or BASP individually. While the lowest concentration (1.2 mgyml) of cIgG evaluated in this experiment did not significantly alter lymphocyte proliferation, a similar concentration of cFab, but not cFc, significantly suppressed mitogen-stimulated proliferation as compared to controls. In this experiment, cFc appeared to interfere with BASP mediated suppression of lymphocyte proliferation, causing significantly higher levels of w3HxTdR uptake than observed due to BASP alone. 4. Discussion Reported estimates of circulating chicken IgG vary widely, but IgG concentrations of 8.7 mgyml have been reported (Yasuda et al., 1998). Thus, it is probable that lymphocytes in the circulation are exposed to IgG concentrations nearly as high as the highest concentrations evaluated in these studies (12 mgyml, Table 5). However, it is much more difficult to estimate the actual exposure of developing neonatal B-lymphocytes to either free IgG, IgG breakdown products, or surface-bound IgG on neighboring lymphocytes within the bursal stroma. Nevertheless, there is likely some exposure to each of these presentations within the developing bursa of Fabricius, as IgG positive lymphocytes have been demonstrated in the bursa prior to hatch (Tao-Wiedmann et al., 1975), and maternally-derived IgG is found in high concentrations in the circulation of day-of-hatch chicks (Zander et al., 1997). The present studies suggest that IgG and some enzymatic degradation products of IgG have anti-proliferative effects on chicken B-lymphocyte proliferation. The effects of IgG and IgG fragments have been widely investigated in human leukocytes. Commercially prepared Human Intravenous Immunoglobulin (IVIG) has been observed to decrease antibody production and B-cell proliferation (Sigman et al., 1998; Kondo et al., 1991). Additionally, IVIG digest products (Fab, F(ab9)2 and Fc) have

also be observed to decrease antibody production and B-cell proliferation (Klaesson et al., 1996; Zhuang and Mazer, 2001). Glick and Olah (1993) have hypothesized that IgG may be required for immature chicken Blymphocyte differentiation and selection within the bursa of Fabricius. In this report, they hypothesized that immature B-lymphocytes, which expressed cell surface IgM, could not differentiate (mature) until they came in contact with IgG, from an unknown source, possibly presented by the bursal secretory dendritic cell (BSDC) in combination with an unknown cytokine, presumably secreted by the BSDC. While preliminary, the present studies may provide some support for the hypothesis of Glick and Olah (1993), suggesting that exogenous IgG can influence proliferation of isolated neonatal B-lymphocytes in birds. It is well known that differentiated cells do not proliferate to the same degree as immature, undifferentiated cells (Freshney, 1983). It can, therefore, be hypothesized that IgG may be acting on these immature neonatal chicken B-lymphocytes to initiate differentiation to more mature cells. However, in human studies, IVIG has been observed to inhibit differentiation of peripheral blood B-lymphocyte (Durandy et al., 1986; Stohl, 1996; Stohl and Elliot, 1996), suggesting that a different mechanism may be involved. Alternatively, it is possible that these results reflect a negative feedback mechanism whereby circulating IgG reduces B-lymphocyte proliferation. This mechanism has been suggested in human lymphocytes by activation of the FcgammaRIIb receptor which decreases B-cell proliferation and activity (Kondo et al., 1991; Ott et al., 2001), but recent data suggest the FcgammaRIIb receptor is not involved in the immunoglobulin associated anti-proliferative activity (Zhuang et al., 2002). Clearly, further studies are required to investigate the role of IgG in lymphocyte proliferation. Interestingly, high concentrations of the Fab fragments (1 mgyml or more) appeared to have a significantly greater anti-proliferative effect than similar molar concentrations of the whole IgG molecule in these studies, with the exception of the 1 mgyml concentration of rFab evaluated in Experiment 3. Additionally, the Fc and F(ab9)2 fragments had effects equal to or greater than the intact IgG molecule (rFc in Experiment 2 and cF(ab9)2 in Experiment 3). This may be due to a higher receptor binding affinity of the IgG frag-

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ments than the whole molecule alone. Although speculative, it is possible that these fragments, which would be expected to exist in relatively low circulating concentrations, might represent a negative feedback mechanism where conditions resulting in high concentrations of intact IgG would have increased total molarity of antibody fragments. In support of this hypothesis, administration IVIG has been observed to reduce B-lymphocyte proliferation and immunoglobulin E production in human lymphocytes (Sigman et al., 1998). Additionally, intact IVIG and Fab, F(ab9)2, and Fc fractions of IVIG have been observed to reduce B-lymphocyte proliferation in vitro (Zhuang and Mazer, 2001). Possible explanations for the observed effects of IgG fragments include a cell membrane receptor site capable of recognizing all IgG fragments, which is possibly in the constant immunoglobulin domains, since it is the most homologous region between all three digest products. However, if a common signal site exists for all digest products, degradation products might have increased effects as compared to intact IgG, as the combined molarity of total effector molecules in vivo would effectively increase through cleavage. As previously stated, this mechanism has been hypothesized in humans, but the involvement of the FcgammaRII receptor is controversial (Zhuang et al., 2002). Alternatively, multiple independent receptor sites on the cell membrane of chicken neonatal B-lymphocytes could exist, which might suggest that the apparently increased effect of high concentrations (1 mgyml or more) of Fab in Experiments 2, 3 (except rFab), and 4 (rFc in Experiment 2, and cF(ab9)2 in Experiment 3) are due to a conformational change or uncovering of binding sites which may occur upon cleavage of the molecule. These hypotheses were not directly tested in the present experiments and investigations specifically addressing these questions are needed. Additionally, the combination of BASP with high concentrations of either whole IgG (2 mgy ml or more) or IgG fragments (0. 65 mgyml or more) appeared to have at least an additive antiproliferative effect in Experiment 4 and in Experiment 2, suggesting an independent mechanism for action of each of the two molecules. Glick and Olah (1993) hypothesized that the BSDC secreted a yet undiscovered substance which, in the presence of IgG, stimulated differentiation of the

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immature B-lymphocytes. Although speculative, the present data may suggest that the observed anti-proliferative effects of BASP are related to initiation of B-lymphocyte differentiation, and that this process is enhanced by the presence of IgG in high concentrations. Further studies to investigate the effects of BASP on chicken B-lymphocytes differentiation are required. Although the effects of immunoglobulins on human lymphocytes have been extensively investigated, the exact mechanism of action remains in question. The present studies are the first to examine the activity of IgG and IgG digest products on avian lymphocytes. The observation that immunoglobulin activity on B-lymphocytes is conserved across both mammalian and avian species suggests that it is an important and fundamental mechanism which needs to be more thoroughly investigated. References Akita, E.M., Nakai, S., 1993. Production and purification of Fab’ fragments from chicken egg yolk immunoglobulin Y (IgY). J. Immunol. Meths. 162, 155–164. Byrd, J.A., Dean, C.E., Fossum, T.W., Hargis, B.M., 1995. Effect of bursal anti-steroidogenic peptide (BASP) on cortisol biosynthesis in ACTH-stimulated canine adrenocortical carcimoma cells in vitro. Vet. Immunol. Immunopath. 47, 35–42. Byrd, J.A., Dean, C.E., Hargis, B.M., 1994. The effect of the humoral immune system derived bursal anti-steroidogenic peptide (BASP) on corticosteroid biosynthesis in avian, porcine and canine adrenal cortical cells. Comp. Biochem. Physiol. 108C, 221–227. Byrd, J.A., Dean, C.E., Hayes, T.K., Wright, M.S., Hargis, B.M., 1993. Detection and partial characterization of an anti-steroidogenic peptide from the humoral immune system of the chicken. Life Sci. 52, 1195–1207. Caldwell, D.J., Dean, C.E., McElroy, A.P., Caldwell, D.Y., Manning, J.G., Hargis, B.M., 1999. Bursal anti-steroidogenic peptide (BASP): modulation of mitogen-stimulated bursallymphocyte DNA synthesis. Comp. Biochem. Physiol. 123A, 385–391. Dean, C.E., Byrd, J.A., Hargis, B.M., 1995a. Bursal antisteroidogenic peptide alters the activity of steroidogenic enzymes in chicken granulosa cells. Domestic Anim. Endocr. 12, 51–61. Dean, C.E., Byrd, J.A., Williams, J.D., Hargis, B.M., 1995b. Influence of follicular maturation on inhibition of luteinizing hormone-, cyclic 39,59-adenosine monophosphate-, and forskolin-stimulated progesterone production in chicken ovarian granulosa cells exposed to bursal anti-steroidogenic peptide. Biol. Reprod. 52, 771–775. Durandy, A., Fischer, A., Griscelli, C., 1986. Dysfunctions of pokeweed mitogen-stimulated T and B lymphocyte responses induced by gammaglobulin therapy. J. Clin. Invest. 67, 867–877.

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immunoglobulin for intravenous use. J. Immunol. 136, 4407–4413. Stohl, W., Elliot, J.E., 1996. In vitro inhibition by intravenous immunoglobulin of human T cell-dependent B cell differentiation induced by staphylococcal superantigens. Clin. Immunol. Immunopathol. 79, 122–133. Tao-Wiedmann, T.W., Loor, F., Hagg, L.B., 1975. Development of surface immunoglobulins in the chicken. Immunology 28, 821–830. Yasuda, M., Furusawa, S., Matsuda, H., Taura, Y., Urano, T., Yokomizo, Y., Ekino, S., 1998. Development of maternal IgG-free chick obtained from surgically bursectomized hen. Comp. Immunonol. Microbiol. Infec. Dis. 21, 191–200. Zander, D.V., Bermudez, A.J., Mallinson, E.T., 1997. Principles of disease prevention: diagnosis and control. In: Calnek, B.W. (Ed.), Diseases of Poultry. 10th ed.. Iowa State University Press, Ames, IA, pp. 1–45. Zhuang, Q., Bisotto, S., Fixman, E.D., Mazer, B., 2002. Suppression of IL-4- and CD40-induced B-lymphocyte activation by intravenous immunoglobulin is not mediated through the inhibitory IgG receptor FcgammaRIIb. J. Allergy Clin. Immunol. 110, 480–483. Zhuang, Q., Mazer, B., 2001. Inhibition of IgE production in vitro by intact and fragmented intravenous immunoglobulin. J. Allergy Clin. Immunol. 108, 229–234.