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Originally published online as doi:10.1189/jlb.1204701 on March 14, 2005

Published online before print March 14, 2005
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(Journal of Leukocyte Biology. 2005;77:923-933.)
© 2005 by Society for Leukocyte Biology

Fibrocytes are potent stimulators of anti-virus cytotoxic T cells

Carole Balmelli, Nicolas Ruggli, Kenneth McCullough and Artur Summerfield1

Institute of Virology and Immunoprophylaxis, Mittelhäusern, Switzerland

1Correspondence: Institute of Virology and Immunoprophylaxis, Sensemattstrasse 293, CH-3147 Mittelhäusern, Switzerland. E-mail: Artur.Summerfield{at}ivi.admin.ch


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Fibrocytes (Fb) are a population of circulating leukocytes reported to be capable of presenting antigen to CD4+ T lymphocytes. In contrast, no information is available about their capacity to stimulate CD8+ cytolytic T lymphocyte (CTL) responses. To this end, Fb were isolated from porcine blood to investigate their ability to stimulate CTL responses using a classical swine fever virus model. The isolated Fb (referred to as primary Fb) displayed the phenotype previously reported for mouse and human Fb, particularly in terms of the surface proteins necessary for antigen presentation, major histocompatibility complex (MHC) classes I and II, and CD80/86. These primary Fb endocytosed and degraded antigen efficiently. In absence of exogenous stimuli, endocytosis and MHC II expression were lost when the Fb were passaged and cultured. Treatment of such secondary Fb with interferon-{gamma} (IFN-{gamma}) restored the MHC II expression. The primary and secondary Fb were capable of stimulating antigen-specific CD4+ T lymphocytes relating to previous reports. In addition, an efficient stimulation of virus-specific CD8+CTL was measured in terms of CD8+ T cell proliferation, IFN-{gamma} production, and cytotoxic activity. This was noted even at low Fb/T lymphocyte ratios, at which dendritic cells were less efficient. Although IFN-{gamma} pretreatment of Fb was not necessary for this function, it could enhance the Fb activity. These results demonstrate that Fb are efficient, accessory cells for the presentation of viral antigen to specific CD8+ CTL.

Key Words: antigen presentating cells • cellular activation • CD8+ T lymphocytes • swine


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Fibrocytes (Fb) are a blood-borne cell population, which has been described in mouse, human, and macaques [1 2 3 ]. They represent 0.5–1% of nucleated cells in peripheral blood and express the hematopoietic/leukocyte cell-surface marker CD34, as well as the fibroblast product collagen I (Col I). In addition, Fb express the major histocompatibility complex (MHC) classes I and II and the costimulatory molecules CD80 and CD86. The precise origin of these cells remains uncertain. Recently, it was shown that Fb can differentiate in vitro from a blood-derived CD14+ population, a process requiring direct T cell interaction [4 ].

Fb rapidly enter sites of injury at the same time as circulating, inflammatory cells. They have been described to be an important source of cytokines and Col I during the inflammatory and repair phases of wound-healing responses [5 ]. Fb are an abundant source of chemokines such as macrophage-inflammatory protein (MIP)-1{alpha}, MIP-1ß, and monocyte chemoattractant protein (MCP)-1, all potent T cell chemoattractants [2 ]. They can produce cytokines such as interleukin (IL)-6, IL-10, and macrophage-colony stimulating factor (M-CSF). Several studies suggested that Fb may play an early role in the induction of antigen-specific immunity [5 , 6 ], and primary Fb were seen to be potent stimulators of CD4+ T lymphocytes [2 ]. Moreover, these cells could stimulate specific immunity in situ [2 ].

The above study focused on the induction of CD4+ T lymphocytes. To date, there have been no reports about the capacity of Fb to induce cytotoxic T lymphocyte (CTL) responses. Consequently, the present investigation sought to determine the capacity of Fb to stimulate CTL responses using a classical swine fever virus (CSFV) model. This virus is a small, enveloped RNA virus of the Flaviviridae, closely related to hepatitis C and Dengue viruses, causing a severe and lethal disease in pigs [7 ]. Our results demonstrated for the first time that Fb are indeed potent accessory cells for the induction of specific antiviral immunity, particularly with respect to activating virus-specific CD8+ CTL at a low Fb/T lymphocyte ratio.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals
Specific, pathogen-free, 3-month-aged pigs (Swiss Land race) were infected intranasally with 103 tissue culture infectivity dose 50% (TCID50) recombinant CSFV Alfort strain [8 ]. All pigs developed clinical disease but recovered 2 weeks postinfection and cleared the virus.

Antibodies
{alpha}-MHC I (clone PT85A) and {alpha}-MHC II (TH16B) monoclonal antibodies (mAb) were purchased from Veterinary Medical Research & Development, Inc. (Pullman, WA). Hybridomas for the mAb {alpha}-swine workshop cluster 3 (SWC3; 74-22-15), {alpha}-CD1 (76-7-4), {alpha}-CD4 (74-12-4), {alpha}-CD8 (11/295/33), {alpha}-CD6 (a38b2), and {alpha}-CD25 (K231.3B2) were kindly provided by Dr. Armin Saalmüller (Federal Research Centre for Virus Diseases of Animals, Tübingen, Germany). The {alpha}-SWC8 (MIL3) hybridoma was kindly provided by Dr. Karin Haverson (University of Bristol, UK). {alpha}-CD16 (G7) mAb was purchased from Southern Biotechnology Associates (Birmingham, AL), {alpha}-CD14 (MY4) from Beckman-Coulter (Nyon, Switzerland), and {alpha}-Col I (mAb-1) from Oncogene (Darmstadt, Germany). CD80/86 expression was detected with recombinant human cytotoxic T lymphocyte antigen-4 mouse immunoglobulin (Ig) fusion protein (Alexis Corporation, Switzerland) as described previously [9 ]. {alpha}-NS3 (C16) mAb was kindly provided by Dr. Irene Greiser-Wilke (Hannover Veterinary School, Germany) [10 ] and {alpha}-E2 mAb (HC/TC26) was obtained from Dr. Bommeli AG (Berne, Switzerland) [10 ]. Reaction of the mAb was revealed using isotype-specific phycoerythrin or biotin-conjugated anti-mouse Ig F(ab')2 fragments (Southern Biotechnology Associates). Biotinylated conjugates were developed with streptavidin-cyanine 5.5 (Dako, Glostrup, Denmark) or streptavidin-Alexa 488 (Molecular Probes, Leiden, The Netherlands). Cells were stained as described previously [9 ]. Data were acquired using a FACSCalibur and analyzed using CellQuest Pro software (BD Biosciences, Mountain View, CA).

Cytokines
Granulocyte macrophage (GM)-CSF was kindly provided by Dr. Shigeki Inumaru (Institute for Animal Health, Ibaraki, Japan) [11 ]. IL-1ß and tumor necrosis factor {alpha} (TNF-{alpha}) were purchased from Endogen (Pierce Biotechnology, Rockford, IL) and transforming growth factor (TGF)-ß1 from R&D Systems (Minneapolis, MN). Interferon-{gamma} (IFN-{gamma}) was kindly provided by Novartis (Basel, Switzerland).

Recombinant porcine IFN-{alpha} (rpIFN-{alpha}) was produced by transient expression in human embryo kidney (HEK)293 Epstein-Barr virus nuclear antigen (EBNA) cells (kindly provided by Dr. Florian Wurm, Federal Polytechnic School of Lausanne, Switzerland) [12 ]. For this purpose, the porcine IFN-{alpha}1 gene was cloned into a mammalian expression plasmid pEAK8 (Edge BioSystems, Gaithersburg, MD). Briefly, peripheral blood mononuclear cell (PBMC)-derived monocytes were stimulated for 18 h with 10 µg/ml polyinosinic polycytidylic acid (Sigma Chemical Co., Buchs, Switzerland) prior to mRNA extraction with the RNeasy kit (Qiagen, Basel, Switzerland). Total RNA was used to synthesize cDNA using an oligo-dT primer and the Omniscript RT kit (Qiagen). The IFN-{alpha}1 gene was amplified from cDNA by polymerase chain reaction (PCR) with the sense primer 5'-GAGAGCAGGGTCACAGAGTCA-3' and the antisense primer 5'-GGAAGAATGGGCTTGTTAGTCT-3', using pfu polymerase (StrataGen, Kirkland, WA). The polymerase chain reaction (PCR) product was cloned in plasmid pCR®-TOPO-XL (Invitrogen, Basel, Switzerland), and six independent clones were sequenced using the Global IR2 system and e-Seq software (LI-COR, Lincoln, NE). The sequence of one clone containing the consensus sequence of the six clones was deposited to GenBank and obtained the accession number AY345969. The cloned IFN-{alpha}1 open-reading frame was then amplified by PCR with the pfu polymerase using the sense primer 5'-CTACCTAAGCTTCACCATGGCCCCAACCTCAGCCTTCCT-3' and the antisense primer 5'-TGCCTCTAGACTCCTTCTTCCTGAGTCTGTCTTG-3' to introduce a HindIII site and a Kozak consensus sequence upstream of the start codon and an XbaI site at the 3' end of the gene, deleting the stop codon. This fragment was then cloned into the HindIII and the XbaI sites of pEAK8-His, constructed by inserting a cassette carrying an XbaI site, the codons for six histidine residues, and a stop codon between the EcoRI and NotI sites of pEAK8 (Michael Horn, unpublished results), resulting in a fusion of the IFN-{alpha}1 gene with the 3'-terminal sequence coding for six histidine residues. His-tagged rpIFN-{alpha}1 was then produced in HEK293 EBNA cells using calcium-phosphate transfection as described [12 ]. The IFN-{alpha}1 bioactivity in the supernatant of the transfected HEK293 EBNA cells was quantified and normalized with commercial rpIFN-{alpha} (R&D Systems) using an Mx/chloramphenicol acetyltransferase reporter gene assay kindly provided by Dr. Martin D. Fray (Institute of Animal Health, Compton, Newbury, Berkshire, UK) [13 ], which was adapted for porcine IFN-{alpha}/ß [14 ].

Fb isolation
Porcine Fb were derived and cultured as described for human [1 ] with some modifications. Briefly, PBMC were isolated from the blood of pigs by density gradient centrifugation over Ficoll-paque (1.077 g/l, Amersham Pharmacia Biotech, AG, Dubendorf, Switzerland) [15 ]. Cells were plated at 4 x 106 cells/ml in Dulbecco’s modified Eagle’s medium (DMEM), 20% (v/v) fetal bovine serum [FBS; 1% (v/v) penicillin/streptomycin (Fb medium, all from Invitrogen] in plastic, 150 cm2 flasks. After 24 h of culture at 39°C, 6% (v/v) CO2, nonadherent cells were removed by a single, gentle aspiration. Following 10–12 days of further continuous culture, growing Fb, which are loosely adherent, were detached from the flasks by a 2-min incubation with cold phosphate-buffered saline (PBS), 0.05% (w/v) EDTA. These were referred to as "primary Fb," as they had never been subcultured and were used immediately or transferred to a 75-cm2 flask for further culture in Fb medium to give "secondary Fb."

Dendritic cells (DC) generation
IFN-{alpha}-derived DC were prepared as described previously [16 ]. Monocytes were obtained by SWC3+ cells, sorting from total PBMC, using magnetic antibody cell sorting (MACS; Miltenyi Biotech, Gladbach, Germany). The resulting cell population was >97% SWC3+. Cells were plated in 24-well plates at a concentration of 1 x 106 cells/ml in DMEM (Invitrogen) containing 10% (v/v) porcine serum (Sigma Chemical Co.), 1% (v/v) penicillin/streptomycin (Invitrogen), GM-CSF (100 U/ml), and rpIFN-{alpha} (1000 U/ml, see above) or rpIL-4 (100 U/ml; DC medium). After 3 days of culture, nonadherent and loosely adherent cells were collected and used in subsequent experiments.

Virus propagation
CSFV (Eystrup strain) was propagated in swine epithelial kidney (SK)-6 cells at a multiplicity of infection (MOI) of 0.01 TCID50/cell for 72 h at 39°C. Intracellular virus was released by sonicating the cells three times for 20 s in 5 ml SK-6 medium [DMEM with Earle’s salts, 7% (v/v) horse serum]. Lysates were then clarified at 3000 rpm for 20 min, and supernatant was aliquoted and frozen at –70°C. For mock controls, clarified lysates of uninfected cells were used. Virus titers were determined by end-point dilution titrations on SK-6 cells as described previously [17 ].

Confocal microscopy
For virus detection, 104 secondary Fb were plated in Labtek plates (Nunc, Life Technologies, Paisley, UK) and infected with CSFV at a MOI of 0.1 TCID50/cell for 72 h. Cells were washed twice with PBS, fixed with 4% (w/v) paraformaldehyde (PFA) for 10 min at room temperature (RT), and washed twice with PBS. {alpha}-NS3 and {alpha}-E2 mAb were added in permeabilization buffer [0.3% (w/v) saponin (Sigma Chemical Co., ref. 32692411 593) in PBS] at 1 µg/test, 30 min at 4°C. For Col I detection, Fb were plated in fibronectin-coated slides and fixed with PFA, and {alpha}-Col I mAb was added in permeabilization buffer at 2 µg/test. Cells were washed with saponin buffer [0.1% (w/v) saponin in PBS] and then incubated for 30 min at 4°C with isotope-specific, Alexa-conjugated, anti-mouse Ig, diluted 1/200 in permeabilization buffer and Alexa 546-phalloidin (5 µg/ml, Molecular Probes). Cells were washed with saponin buffer and with PBS before mounting and viewing with a Leica TCS-SL spectral confocal microscope equipped with Leica LCS software (Leica Microsystems AG, Glattbrugg, Switzerland).

Endocytic activity
Endocytic activity of primary and secondary Fb was measured using DQ-BODIPY-labeled bovine serum albumin (BSA-DQ; Molecular Probes), a reagent heavily labeled with a fluorescent dye, resulting in the total quenching of the fluorescence. Upon uptake and endocytic proteolysis, the protein is cleaved, thus removing the quenching and permitting detection of the fluorescent signal. Fb (2.5x105) were incubated with 10 or 1 µg/ml BSA-DQ for 30 min at 39°C or 4°C in DMEM without serum. Cells were washed twice with cold PBS and analyzed by flow cytometry.

Antigen presentation assay
Primary Fb were detached with PBS-EDTA and resuspended at 2 x 105/ml in complete medium containing 20 µM 2-mercaptoethanol (2-ME; Invitrogen) and 1 µg/ml mitomycin C (Sigma Chemical Co.). After 16 h incubation, the plates were washed five times with DMEM 2% (v/v) FBS. Secondary Fb were treated or not with 100 ng/ml IFN-{gamma} for 72 h before harvest. Cells were detached from the flask surface with trypsin-EDTA (Invitrogen), washed in 10 ml DMEM 2% (v/v) FBS, and resuspended at 2 x 105/ml in complete medium containing 20 µM 2-ME (Invitrogen). Purified lymphocytes were prepared from PBMC by immunomagnetic depletion of the SWC3+ myeloid mononuclear cells containing the DC and the monocytes and blood DC [18 ], using MACS. Briefly, PBMC were incubated with mAb for 20 min on ice and washed once with cold MACS buffer [PBS EDTA, 1% (v/v) FBS]. Cells were then incubated with isotype-specific MACS Ig microbeads (Miltenyi Biotech) for 15 min on ice. After washing with cold PBS, cells were resuspended in 2 ml MACS buffer and sorted on a MACS LD column (Miltenyi Biotech). Purity of the negative fraction was over 98%. For the lymphoproliferation assays, 2.5 x 105 sorted lymphocytes were incubated with autologuous primary or secondary Fb in 96-well, flat-bottom plates at various Fb/lymphocyte ratios in triplicate wells in the presence of CSFV (MOI of 0.01 TCID50/cell), ultraviolet (UV)-inactivated CSFV (MOI of 1 TCID50/cell), or mock antigen. The MOI was calculated on the basis of the total number of cells (Fb and lymphocytes), and the virus was added when the Fb and the lymphocytes were brought into coculture. For blocking experiments, 50 µl hybridoma supernatant containing {alpha}-CD4 (74-12-4), {alpha}-CD8 (11/295/33), or {alpha}-CD1 (76-7-4) was added to the coculture 1 h prior to the addition of virus. The proliferative activity was measured after 5 days by addition of 1 µCi 3H-thymidine/well for the last 16 h of culture.

Cross-presentation assays
For cross-presentation using infected apoptotic (APO) cells, Fb were incubated with UV-inactivated CSFV (104J/m2, 20 min) at a MOI of 100 TCID50/cell for 2 h before the assay in 50 µl total volume. This MOI was calculated from the virus titer before inactivation, and inactivation of the virus was controlled by passage on SK-6 cells, which were then seeded into 24-well plates at a concentration of 5 x 105 cells/ml and UV-irradiated with 104J/m2 for 20 min to induce APO Fb. Secondary Fb were mixed with 1.5 x 107 immune lymphocytes (prepared as described above) at a ratio or 1/400 in a final volume of 3 ml complete medium. APO Fb were added to these cocultures at an estimated Fb/APO Fb ratio of 1:3. For cross-presentation using exogenous antigen, CSFV was grown in SK-6 cells for 3 days at 39°C. The infected cells were lysed by sonication (see above) and then treated with UV (104 J/m2, 20 min) to inactivate the virus present. This material was added to the Fb/lymphocyte coculture (1/400 ratio, prepared as described above) at a MOI of 2 TCID50/cell, calculated from the virus titer before inactivation.

Stimulation of CSFV-specific CTL and IFN-{gamma}-producing cells
Autologous, secondary Fb were pretreated or not with IFN-{gamma} and mixed with 1.5 x 107 lymphocytes (prepared as described above) at a ratio of 1/400 in a final volume of 3 ml complete medium. CSFV was added at a MOI of 0.01 TCID50/cell, and the cocultures were incubated for 5 days before testing for the presence of activated T cells by enzyme-linked immunospot (ELISPOT) assay, cytotoxic assay, or flow cytometry (see below).

IFN-{gamma} ELISPOT
The ELISPOT assay was performed as described elsewhere [19 ] with minor modifications. Different concentrations of stimulated lymphocytes were added to duplicate wells on nitrocellulose plates coated with mouse anti-pig IFN-{gamma} mAb (MP700, 5 µg/ml, Pierce Biotechnology). After 48 h incubation, IFN-{gamma} spots were visualized with rabbit anti-pig polyclonal antibody (PP700, 2.5 µg/ml, Pierce Biotechnology) followed by goat anti-rabbit peroxidase-conjugated antibody (Jackson Immunoresearch Laboratories, West Grove, PA) and developed with 3,3'-diaminobenzidine (Sigma Chemical Co.) for 20 min at RT in the dark. The number of spots was determined with a computer-assisted video image analyzer (ELISPOT reader, AID GmbH, Strassberg, Germany).

Cytokine assay
Cell-free supernatant from the Fb/lymphocyte cocultures was tested for the presence of IFN-{gamma}, IL-10, and IL-4 using enzyme-linked immunosorbent assay (ELISA) kits (all from Biosource, Camarillo, CA). The detection limits were 7.5 pg/ml for IFN-{gamma} and IL-10 and 15.6 pg/ml for IL-4.

Triple-color flow cytometry
Discrimination of T cell subpopulations was performed as described previously [20 ] with some modifications. Briefly, stimulated lymphocytes were harvested, counted, and labeled simultaneously with {alpha}-CD4, {alpha}-CD8, and {alpha}-CD25. Gating of living cells was performed as described elsewhere [21 ]. This permitted the identification of CTL cells CD4–CD8highCD25high. The absolute number of CD4–CD8highCD25high (x106/ml) among stimulated lymphocytes was calculated as follows: absolute number of mononuclear cells (calculated from the stimulated lymphocytes) x percentage of positive cells (CD4–CD8highCD25high) in the mononuclear gate:100. The ratio of CD4–CD8highCD25high in a total of 106 lymphocytes was then calculated: number of CD4–CD8highCD25high/106 total mononuclear harvested cells.

Cytotoxic assay
Autologous Fb infected with CSFV were used as target cells in a flow cytometry assay for cytotoxicity [22 ]. Briefly, Fb were detached, stained with PKH26, according to the manufacturer’s instructions (Sigma Chemical Co.), and plated at 10,000 cells/well in 96-well, flat-bottomed plates. Then, cells were infected with CSFV or treated with mock antigen for 24 h at a MOI of 2 TCID50/cell. Stimulated lymphocytes were sorted by MACS with the {alpha}-CD6 mAb as described previously [9 ] to eliminate unspecific killing by natural killer cells [23 ] and added to the target Fb at various effector:target (E:T) ratios. After overnight incubation at 39°C, all cells were collected using trypsin-EDTA and resuspended in 150 µl PBS. Immediately before cytometric analysis, 0.1 µg propidium iodide (PI; Sigma Chemical Co.) was added. At least 3000 PKH26high cells were acquired for each condition.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Morphology and phenotype of porcine Fb
Primary Fb, obtained after the 10–12 days of PBMC culture described in Materials and Methods, appeared as elongated, spindle-shaped cells [1 , 4 ]. No cell purification was necessary, as these primary Fb detached rapidly in PBS-EDTA. In contrast, the other adherent cells with macrophage morphology remained attached. From 100 x 106 PBMC seeded, 0.5–1.5 x 106 were recovered after 11 days of culture. The passaged Fb readhered readily and upon culture, displayed the typical spindle-shaped morphology reported for human and mouse Fb (see Fig. 3 ). These cells increase in size, show a reduced doubling rate, and form a confluent monolayer within 1 week. They displayed extensive stress fiber formation (see Fig. 3 ), which distinguished them from monocytes, macrophages, and DC. Such passaged and cultured Fb were termed secondary Fb.



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  		Figure 3. CSFV infection and replication in Fb. (A) Cultured (secondary) Fb were infected (A, B, E, and F) or mock-treated (C, D, G, and H) at a MOI of 0.1 TCID50/cell for 3 days. After fixation, the cells were stained with mAb against the viral nonstructural protein NS3 (B and D) or the virion glycoprotein E2 (F and H). (B) Primary or secondary Fb were plated on a fibronectin-coated chamber slide. After fixation, the cells were stained with the mAb against Col I (II and IV). The spindle-shaped morphology of the Fb is shown by staining the microfilaments and stress fibers with Alexa 546-conjugated phalloidin (A, C, E, G, I, and III).

 
Primary and secondary peripheral blood-derived Fb were characterized phenotypically with respect to myeloid markers and molecules related to antigen presentation. The primary Fb expressed high levels of MHC II, MHC I, CD80/86, and CD16, as well as CD1 (Fig. 1A ). In addition, the porcine SWC3 marker, equivalent to human CD172, expressed mainly on myeloid cells [24 ], was also expressed at a high level on most primary Fb. In contrast, CD14 and SWC8, markers found on all granulocytes, all B cells, and many T cells but not on monocytes and macrophages, were only expressed on subpopulations of the primary Fb. The fibroblast product Col I expression was detected on the cell surface as reported for human Fb [4 ] but at low levels. Expression of Col I by primary Fb was confirmed by confocal microscopy, demonstrating high levels of intracellular Col I (see Fig. 3B , I and II).



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  		Figure 1. Phenotype of Fb obtained from an 11-day culture of primary Fb (A) and secondary Fb maintained in culture for 1 month after three passages (B) analyzed by forward-scatter (FSC) and side-scatter (SSC; dot-plots), as well as the expression of particular cell-surface markers (histograms) as shown. Unfilled histograms show conjugate controls. A representative experiment out of three is shown.

 
On secondary Fb, the expression of MHC II, CD14, CD16, and CD1 became undetectable after 2 additional weeks of culture (Fig. 1B) . In contrast, MHC I, CD80/86, and SWC3 were still expressed, although the intensity of expression for the latter two molecules was clearly diminished compared with that on primary Fb. Although Col I was no more expressed on the cell surface of secondary Fb, high levels of intracellular Col I were still detected (see Fig. 3B , III and IV). It is interesting that all secondary Fb now expressed the SWC8. It was possible that the secondary Fb resulted from an outgrowth of the SWC8+ population, existing among the primary Fb. Consequently, the primary Fb were sorted into SWC8+ and SWC8– fractions. Purity of the SWC8– fraction was >99%. The SWC8+ and SWC8– population fractions of primary Fb were then cultured separately. Similar growth of the Fb was noted with both cultures. Furthermore, testing for CD14 and SWC8 expression revealed that both cultures expressed SWC8 and CD14, indicating an up-regulation of SWC8 on all Fb during culture.

Response of Fb to inflammatory cytokines
The inflammatory cytokines TNF-{alpha}, IL-1ß, and TGF-ß1 are present at the site of wound-healing and implicated in the stimulation of fibrogenic responses [25 ]. Fb can respond to IL-1ß in terms of proliferation [5 ] and to TGF-ß1 in terms of phenotypic modulation [4 , 26 ]. In the present study, the influence of TNF-{alpha}, IL-1ß, and TGF-ß1 on the expression of accessory molecules was investigated. In addition, the influence of IFN-{gamma} was analyzed. All these cytokines, with the exception of TGF-ß1, promoted secondary Fb proliferation, seen as a doubling of division rate (data not shown). TGF-ß1 also had no detectable effect on accessory molecule expression (data not shown), whereas IFN-{gamma}, TNF-{alpha}, and IL-1ß induced an up-regulation of MHC I expression on the Fb (Fig. 2A , upper row). In contrast, neither TNF-{alpha} nor IL-1ß influenced expression of MHC II (Fig. 2A , lower row). There was also no influence on CD80/86 or CD16 expression (data not shown). IFN-{gamma} was the only cytokine that modulated MHC II levels (Fig. 2A , lower row). The IFN-{gamma}-induced up-regulation of MHC II displayed a slow kinetic, reaching maximum at 72 h after stimulation.



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  		Figure 2. (A) Phenotypic modulation of Fb stimulated with inflammatory cytokines. Secondary Fb were stimulated (filled histograms) for 72 h with 100 ng/ml of the indicated cytokine and stained for MHC I (upper panel) or MHC II (lower panel) expression. Empty histograms show untreated cells. A representative experiment out of two is shown. (B and C) Endocytic activity of Fb and DC. (B) Primary Fb, IL-4/GM-CSF DC, or (C) secondary Fb, pretreated or not with IFN-{gamma} (100 ng/ml, 72 h, last histogram), were incubated with 1 µg or 10 µg/ml BSA-DQ for 30 min at 4°C (empty histograms) or 39°C (filled histograms).

 
Endocytic activity of Fb
Although primary Fb have been reported as inducers of antigen-specific proliferation [2 ], there is no information about their efficiency of antigen uptake and processing. The primary Fb in the present report were seen to be efficient at endocytosis and degradation of BSA-DQ (Fig. 2B) . A high signal was obtained as early as 30 min after incubation of the Fb with 1 µg/ml BSA-DQ. The signal intensity increased with 10 µg/ml BSA-DQ, demonstrating that the Fb endocytosis was not saturated by 1 µg/ml and also the high endocytic potential of these cells. These characteristics are similar to those seen following endocytosis of BSA-DQ by IL-4/GM-CSF-derived DC (see Fig. 2B , last histogram and ref. [18 ]). In contrast, secondary Fb displayed a relatively low endocytic activity (Fig. 2C) . Only a weak fluorescent signal was obtained and then only with 10 µg/ml BSA-DQ. It is interesting that treatment of the secondary Fb with IFN-{gamma} did not enhance this endocytic activity (Fig. 2C , last histogram).

Fb susceptibility to virus infection
As a result of the affinity of CSFV for myeloid cells [17 , 27 ], this virus was chosen as a model to determine the susceptibility of secondary Fb to virus infection. At 72 h post-infection (MOI of 0.1 TCID50/cell), the viral structural protein E2 and the nonstructural protein NS3 were detectable (Fig. 3A ). NS3 showed the typical perinuclear staining, and E2 was more diffuse in the cytoplasm. The results demonstrated that Fb are susceptible to a productive infection by CSFV (the viral detection method does not show virus entry but only de novo synthesized proteins). It is important that the virus did not induce morphological changes in the cell, witnessed by the unaltered staining pattern of the microfilament stress fibers (Fig. 3A) . Comparable susceptibility to CSFV infection was observed for primary Fb (data not shown) and with monocyte-derived DC (MoDC) [28 ].

Role of Fb in a virus-specific, proliferative response
Fb have been reported to induce specific proliferative responses against exogenous soluble antigen [2 ]. It was considered necessary to determine the efficiency of the CSFV-infected Fb in this activity, and the CSFV infection provided more endogenous antigen. Consequently, virus-infected secondary Fb (MOI of 0.01 TCID50/cell) were incubated for 5 days with autologous lymphocytes from a CSFV-immune pig at the ratios shown in Figure 4 . As a result of the results obtained with MHC II expression on secondary Fb (see Fig. 2 ), the CSFV-infected Fb were treated with IFN-{gamma} for 72 h prior to the coculture with the immune lymphocytes. The Fb were infected with the virus or treated with mock antigen at the time of adding the immune lymphocytes for coculture. When Fb were pretreated with IFN-{gamma}, this had no influence on the characteristics of the virus infection in these cells (data not shown).



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  		Figure 4. Fb-dependent, virus-specific T cell proliferation. (A) Capacity of CSFV-infected Fb to stimulate CSFV-specific proliferation. Lymphocytes (2.5x105) from a CSFV-immune pig (Ly+CSFV) were cocultured at various ratios with autologous Fb (x-axis) infected at the time of coculture (MOI of 0.01 TCID50/cell) or mock-treated (open bars). (A) The Fb were treated or not for 72 h with IFN-{gamma} (100 ng/ml, solid bars and dark-shaded bars) prior to coculture. (B) Cocultures of IFN-{gamma}-treated Fb and autologous immune lymphocytes were infected at the time of coculture with CSFV (solid bars, MOI of 0.01 TCID50/cell) or mock-treated (open bars). In certain cultures, anti-CD8 (hatched bars) or anti-CD4 (shaded bars) were added at the beginning of the coculture. After 4 days of incubation, the cultures were pulsed for 12 h with 1 µCi 3H-thymidine. (C) CSFV-specific proliferation induced by primary Fb. The conditions were as those in B, except there was no IFN-{gamma} pretreatment. CPM, Counts per minute.

 
Even at low Fb/lymphocyte ratios, virus-specific proliferation was observed (Fig. 4 , A and B, solid bars, CSFV; open bars, mock). The IFN-{gamma} pretreatment of the Fb did increase the induced lymphoproliferation but mainly at the higher Fb/lymphocyte ratios (Fig. 4A , solid compared with dark-shaded bars). No specific proliferation was observed in the absence of Fb or when using lymphocytes from a naive pig; the results in these cases were similar to those with the mock-infected cultures shown in Figure 4 , A and B. Virus-specific proliferation was obtained with UV-inactivated virus (MOI of 1 TCID50/cell tested before inactivation), attesting to the capacity of Fb to take up, process, and present inactivated viral antigen (data not shown).

The virus-infected, Fb-induced immune response was suppressed in the presence of saturating concentrations of {alpha}-CD8 mAb at all Fb/lymphocyte ratios (Fig. 4B , hatched bars). Saturating concentrations of {alpha}-CD4 mAb only affected proliferation at the highest Fb/lymphocyte ratio (1/25, Fig. 4B , shaded bars). Identical results were obtained when {alpha}-MHC II (MSA3) was used for blocking. The blocking of CD4-dependent T cell activation using the clone 74-12-4 and of CD8-dependent T cell activation using the clone 76-2-11 was based on the study of Pescovitz et al. [29 ]. The use of these mAb for blocking of MHC I- and II-restricted T cell activation is well-established in porcine immunology [30 31 32 ]. An anti-CD1 (IgG2a) was used as a control antibody for the blocking experiment, and no effect was observed (data not shown). These data showing a CD4 and CD8 dependency of the virus-infected Fb stimulation of lymphocyte proliferation had been obtained using secondary Fb. It is important that similar results were obtained with primary Fb (Fig. 4C) . Virus-specific proliferation was observed at low Fb/lymphocyte ratios, and proliferation was blocked with {alpha}-CD8 mAb. Again, no effect of {alpha}-CD1 was observed (data not shown). This demonstrates that despite the phenotypic modulations, secondary Fb retained the capacity of primary Fb to stimulate CD8+ lymphocytes. It is interesting that this stimulation could be obtained using fresh or cryopreserved lymphocytes (Fig. 4C) .

Fb stimulation of CTL activity
To elaborate further on the Fb stimulation of CD8-dependent responses, CSFV-infected (MOI of 0.01 TCID50/cell) and mock-treated Fb were cocultured with CSFV-immune lymphocytes at a ratio of 1/400. As additional controls, lymphocytes alone were infected with the virus. After 5 days of incubation, the Fb were examined by microscopy, and the lymphocytes were tested for the presence of activated T cells by IFN-{gamma} ELISPOT assay. Although CSFV infection did not alter Fb morphology (Fig. 3) , this was not the case when lymphocytes from a CSFV-immune animal were present. Although Fb from mock or untreated coculture showed typical adherent, spindle-shaped morphology (Fig. 5A ), few adherent Fb remained in the infected coculture (Fig. 5B and 5C) . Moreover, the cytoplasmic mass appeared to be reduced in these latter Fb, and they were surrounded by lymphocytes (confirmed by confocal microscopy and labeling of the Fb and lymphocytes). This morphological change and disappearance of Fb were only observed in the cocultures, not when CSFV-infected Fb were grown in the absence of lymphocytes, and suggest the presence of a CTL-mediated killing activity in the infected cocultures.



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  		Figure 5. Loss of Fb from cocultures. Secondary Fb and immune lymphocytes were mixed at a ratio of 1/400 and mock-treated (A) or infected with CSFV at a MOI of 0.01 TCID50/cell (B and C). After 5 days, the lymphocytes were removed by washing, and the remaining cells in culture were photographed (original magnification, 100x). (C) A higher original magnification (200x) of the infected coculture in B. A representative experiment out of four is shown.

 
The cocultures of CSFV-immune lymphocytes with virus-infected, secondary Fb yielded high numbers of IFN-{gamma} secreting cells (SC) (Fig. 6A ), not observed with lymphocytes alone or in the absence of CSFV infection. IFN-{gamma} pretreatment of the Fb resulted in a small enhancement of this number, but this was not statistically significant (P>0.01, as determined by Student’s t-test). High levels of IFN-{gamma} were detected in the supernatants of the 5-day cocultures of CSFV-infected Fb plus immune lymphocytes (Fig. 6B) , in contrast to mock-treated cocultures or infected lymphocytes alone. No IFN-{gamma} was detectable in the supernatant of the infected Fb cultured alone (data not shown). When {alpha}-CD8 or {alpha}-CD4 mAb but not {alpha}-CD1 mAb was added at the beginning of the coculture, the IFN-{gamma} was impaired (Fig. 6B) . No IL-4 and only low levels of IL-10 (30 pg/ml) were detected (data not shown). It is interesting that although a consistent titer of virus was present in the supernatant of infected Fb (104.5 TCID50/ml), no virus was detectable in the coculture (data not shown). This was also found in the coculture treated with {alpha}-CD4 and {alpha}-CD8, reflecting the incomplete blocking of the immune response by the antibodies.



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  		Figure 6. Fb-dependent activation of CTL. Secondary Fb, treated or not with IFN-{gamma} (IFN-{gamma} or Untreated, respectively), were incubated at a ratio of 1/400 with autologous lymphocytes. The cocultures were infected with CSFV (MOI of 0.01 TCID50/cell, solid bars) or mock-treated (open bars) at the initiation of the coculture. After 5 days of culture, the lymphocytes were analyzed in an IFN-{gamma} ELISPOT assay (A) and supernatants in an IFN-{gamma} ELISA (B). Infected cultures without Fb ("ly") served as an additional control. Errors bars indicate the SD between replicate wells. A representative experiment out of four is shown. (B) {alpha}-CD8, {alpha}-CD4, or {alpha}-CD1 (200 µl; control antibodies) was added at day 0 and day 1 to certain cocultures to identify the type of T cell response. (C) The cytotoxic activity of the lymphocytes following the 5 days coculture with infected Fb was tested using additional cultures of autologous Fb, which were infected with CSFV (MOI of 2 TCID50/cell) or mock-treated 24 h prior to adding the stimulated lymphocytes. Means of duplicates ± SD are shown. This experiment is representative of three independent cytotoxic experiments.

 
The results from Figure 5 were showing that the presence of immune lymphocytes resulted in damage to the virus-infected Fb. This was related to a cytotoxic immune response by stimulating lymphocytes from an immune animal with CSFV-infected Fb for 5 days and then incubating at various E:T ratios with infected or mock-treated target Fb. A three- to fourfold increase of specific lysis was observed with the infected target cells compared with mock-treated target cells (Fig. 6C) . This confirmed the observations in Figure 5 that lymphocytes stimulated with virus-infected Fb could recognize and lyse virus-infected target Fb specifically.

Comparative efficiency of Fb and DC as stimulators of CD8+ lymphocytes
It was recently shown that GM-CSF/IFN-{alpha}-induced MoDC (IFN-{alpha} DC) were potent activators of CTL activity [33 34 35 36 ]. Consequently, GM-CSF/IFN-{alpha} DC were compared with Fb for their ability to activate CD8+ lymphocytes. The porcine IFN-{alpha} DC exhibited the same morphological and phenotypic characteristics as their human counterparts [35 ]: long and fine dendritic-like process, expression of MHC II, CD80/86, and high levels of MHC I (data not shown). Cocultures of immune lymphocytes with Fb or DC infected at the time of coculture were used at Fb/lymphocytes or DC/lymphocytes ratios of 1/10 and 1/400. Following incubation for 5 days, the presence of CD4–CD8highCD25high-activated lymphocytes (CTL phenotype) [37 ] was analyzed by flow cytometry. Figure 7A shows dot-plots of the blasting CD4– lymphocytes selected through electronic gating. A clear increase of CD4–CD8highCD25high cells was detected in cocultures of immune lymphocytes stimulated with virus-infected Fb (Fig. 7B , plots I and II; Fig. 7C , IFN-{gamma} and untreated, solid bars) compared with mock-treated cocultures (Fig. 7B , plots VI and VII; Fig. 7C , open bars). This response required the Fb, as no such increase was seen with infected lymphocytes alone (Fig. 7B , plots V and X; Fig. 7C , lymphocytes). Only a slight increase of CD4–CD8+CD25high cells was observed when the lymphocytes were cocultured with virus-infected IFN-{alpha} DC at the same low (1/400) ratio used with the Fb/lymphocyte culture (Fig. 7A , plot III; Fig. 7B , DC 1/400). However, when the cocultures used the DC at the 1/10 DC/lymphocyte ratio (Fig. 7B , plot IV; Fig. 7C , DC-IFN-{alpha} 1:10), these cells were efficient activators of the CD4–CD8+CD25high population. As a result of the large size of Fb, such a ratio is not practical for a stimulation assay. However, when the Fb and IFN-{alpha} DC were compared at a ratio of 1/100, a similar capacity to stimulate CD4–CD8+CD25high was observed (data not shown).



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  		Figure 7. Comparison of CTL activation by Fb or by MoDC. (A) FSC/SSC of lymphocytes after 5 days of stimulation with infected or mock-treated autologuous Fb. (B) Fb or IFN-{alpha} DC were cultured at a ratio of 1/400 (I–III and VI–VIII) or 1/10 (panels IV and IX) with autologous, immune lymphocytes for 5 days. Infection with CSFV (MOI of 0.01 TCID50/cell, upper panels) or mock treatment (lower panels) was at the initiation of the coculture. Activated CTL were identified using a triple immunofluorescence analysis as CD4–CD8highCD25high within a gate on blasting cells with high FCS. Infected cocultures without Fb or DC (lymphocytes, panel V) served as additional controls. Percent of CD4–CD8highCD25high within the gate is noted. A representative experiment out of two is shown. (C) The numbers of CD4–CD8highCD25high cells harvested from the cultures shown in A, expressed as per 106 total cells harvested (a total of between 9x106 and 11x106 cells were harvested).

 
Cross-presentation of exogenous antigens by Fb
To elaborate on the role of Fb as an antigen-presenting cell (APC) for MHC I presentation, we tested the ability of Fb to cross-present two forms of antigen: derived from apoptotic cells (APO Fb) containing inactivated CSFV antigen or UV-inactivated, CSFV-infected cell lysates. A clear increase of CD4–CD8highCD25high cells was detected in cocultures stimulated with UV-inactivated virus (Fig. 8A , inact. CSFV). A 10-times higher stimulation was observed when live virus was used (Fig. 8B , live CSFV). In contrast, no stimulation was noted when the Fb/lymphocytes cocultures were fed with APO Fb carrying CSFV antigen (data not shown).



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  		Figure 8. Cross-presentation of exogenous antigens by Fb. Secondary Fb were incubated at a ratio of 1/400 with autologous lymphocytes. The cocultures were then fed with (A) UV-inactivated CSFV (MOI of 2 TCID50/cell, inact. CSFV) or (B) live CSFV (MOI of 0.01 TCID50/cell, live CSFV). Cultures without Fb ("ly") served as an additional control. Activated CTL were identified using triple immunofluorescence analysis as CD4–CD8highCD25high. The numbers of CD4–CD8highCD25high cells harvested from the cultures were expressed as per 106 total cells harvested (a total of between 9x106 and 11x106 cells were harvested). These results are representative of three independent experiments. (C) The cytotoxic activity of the lymphocytes following the 5 days of coculture with Fb and inactivated virus. Means of duplicates ± SD are shown. This experiment is representative of two independent cytotoxic experiments.

 
The responses shown in Figure 8 required the presence of Fb, as no such increase was seen with stimulated lymphocytes alone (Fig. 8A and 8B , inact. CSFV ly and live CSFV ly). Increased lysis of infected target cells was observed with lymphocytes from the cocultures stimulated with inactivated virus (Fig. 8C) . These results demonstrate that lymphocytes stimulated by Fb, fed with inactivated virus, could specifically recognize and lyse virus-infected target Fb, thus revealing the capacity of Fb to present exogenous antigen via the MHC I pathway.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
APC with potent immunostimulatory capacities are the critical targets when developing novel vaccines and immunotherapies against infectious diseases and cancer. Although several cell types have the potential for antigen presentation to stimulate T cells, DC are considered to be most potent in this function [38 ]. In this context, recent attention has been drawn to another population of blood-borne cells, the Fb. Characterized in terms of their role in wound healing [5 ], Fb also display an APC role by inducing strong proliferation of CD4+ T cells. [2 ]. Studies in mouse and human have shown that Fb possess a DC-like phenotype, expressing the pan-myeloid marker CD13 along with MHC II and CD80/86 and are negative for CD14 and CD16 [1 , 2 , 38 39 40 ]. Fb isolated from pig peripheral blood also display DC-like characteristics. They possess a high endocytic activity and express all accessory molecules required for efficient antigen presentation. Their expression of the myeloid marker SWC3 would suggest a relationship to monocytes/macrophages and DC [9 , 41 42 43 ]. Moreover, like porcine MoDC and bone marrow-derived DC [9 ], porcine Fb express CD14, CD16, and CD1. The SWC8 marker was expressed heterogeneously by these primary Fb but up-regulated rapidly on all Fb after passage.

Upon passage and culture of the Fb (secondary Fb), the monocytic cell markers, CD80/86, MHC II, and CD1, as well as endocytic activity, were down-regulated. Moreover, the endocytic activity and the doubling rate were reduced. The cytokines TNF-{alpha}, IL-1ß, and IFN-{gamma} induced an up-regulation of MHC I, but only IFN-{gamma} increased MHC II expression on secondary Fb. IFN-{gamma}-dependent enhancement of MHC molecule expression has been reported for other cell types such as endothelial cells, monocytes, fibroblasts, and macrophages [44 ]. Considering the phenotypes of primary Fb and IFN-{gamma}-treated secondary Fb, it seems likely that the observed down-regulation of cell-surface molecules on secondary Fb was a culture-dependent phenomenon. The genetic capacity to express MHC II molecules, especially in response to IFN-{gamma}, was retained.

Fb stimulation of antigen-specific responses has focused on CD4+ lymphocyte activity [2 ]. In contrast, there is no information about the capacity of Fb to stimulate antigen-specific CTL responses. Primary and secondary Fb infected with CSFV induced proliferation of virus-specific lymphocytes, with a comparable efficiency than IL-4/GM-CSF-derived MoDC [28 ]. This demonstrated that despite the phenotypic modulation of secondary Fb, the cells retained their capacity to stimulate antigen-specific immune responses. A CD4+ cell dependency was noted in these immune responses induced by primary or secondary Fb, relating to that already reported for primary Fb [2 ]. It is interesting that a relatively high Fb/lymphocyte ratio (1/25) was required to stimulate CD4+ lymphocytes. It was considered that this might be reflecting the use of a virus infection in the Fb, producing more endogenous peptide antigens and therefore MHC I epitopes. Indeed, a particularly potent induction of virus-specific CD8+ lymphocytes was observed, and this was detectable even at low Fb/T lymphocyte ratios (1/800).

Cytokine analysis of the cocultures of virus-infected, secondary Fb with immune lymphocytes revealed a T helper cell type 1 (Th1) profile: high levels of IFN-{gamma} but little or no IL-10 and IL-4. The IFN-{gamma} production was sensitive to blocking by {alpha}-CD8 and {alpha}-CD4 mAb, even at the low Fb/lymphocytes ratios, with which only CD8+ cell-dependent proliferation was noted. This would suggest that secondary Fb interacted with CD8+ and CD4+ lymphocytes but not always inducing proliferation. Such results could be reflecting an induction of CD8+ cell expansion of effector cytotoxic cells, aided by IFN-{gamma} induced in existing effector CD4+ cells. Indeed, the virus-infected, secondary Fb were potent inducers of cytotoxic activity, observable at the same low ratio (1/400) as the induced proliferation of CD8+ cells. The IFN-{gamma} production correlated with the high cytotoxic activity and was reflected in the number of IFN-{gamma} SC found in cocultures of infected Fb and immune lymphocytes.

It is not surprising that the infected secondary Fb inducing a cytotoxic response were also capable of functioning as targets. Destruction of infected secondary Fb required interaction with lymphocytes from an immune animal and was not a virus-induced, cytopathic effect. Not only were the infected Fb attacked, but also, the production of infectious virus was affected. Therefore, the lymphocytes were attacking the infected Fb rather than inducing the virus to kill.

GM-CSF/IFN-{alpha}-derived DC promote CTL activation and a Th1 response with a higher efficiency than the more commonly used GM-CSF/IL-4-derived DC [34 ]. When secondary Fb were compared with such "IFN-{alpha} DC," at the 1/400 APC/lymphocytes ratio, the Fb were more efficient at activating CD4–CD8+ lymphocytes. Nevertheless, IFN-{alpha} DC were potent APC at the 1/10 APC/lymphocytes ratio. It is important that Fb were able to present inactivated virus to CD8+ CTL. This relates to the form of cross-presentation using exogenous antigen, as reviewed by Heath et al. [45 ] and Ackerman and Cresswell [46 ]. Thus, Fb can function as specialized APC for class I-restricted antigens. Nevertheless, this picture is complicated by the apparent inability of Fb to cross-present apoptotic cells carrying inactivated CSFV. This may be a result of an absence of receptors involved in the uptake of apoptotic cells, such as CD36 or {alpha}vß3 and/or {alpha}vß5 [47 , 48 ]. Future analyses are designed to determine the receptor expression of Fb compared with DC.

The present report demonstrates for the first time that Fb are potent APC, not only for CD4+ lymphocytes expansion but particularly, for the CD8+ CTL. One reason for this potency of Fb may be related to the reported abundant cytokine and chemoattractant secretion, IL-1ß, TNF-{alpha}, MIP-1{alpha}, MIP-1ß, growth-related oncogene-{alpha}, and MCP-1 [2 , 5 ]. Secretion of IL-1ß and TNF-{alpha} could promote early activation of lymphocytes, which in turn, would secrete IFN-{gamma}, capable of enhancing the antigen presenting activity of the Fb. The reported migratory capacities of Fb into regional lymph nodes [2 ] would be of value in their role as APC. Consequently, Fb may prove to be a potent tool for vaccine and immunotherapy research, especially considering their ease of isolation from the blood and adaptability to in vitro expansion (secondary Fb) while retaining APC functionality.


   ACKNOWLEDGEMENTS
 
This work was supported by the Swiss Federal Office for Education and Science (Project BBW 00.0635 linked to EU project QLRT-200-01374). We thank Heidi Gerber for preparing the mAb and Viviane Neuhaus for preparing of recombinant cytokines. We are particularly indebted to Peter Zulliger and Daniel Brechbühl for care of the animals and the regular bleeding.

Received December 3, 2004; revised January 25, 2005; accepted January 26, 2005.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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