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The Journal of Immunology, 2001, 166: 2937-2943.
Copyright © 2001 by The American Association of Immunologists

Thioredoxin Acts as a B Cell Growth Factor in Channel Catfish1 ,2

Morad Khayat, Tor B. Stuge, Melanie Wilson, Eva Bengtén, Norman W. Miller and L. William Clem3

Department of Microbiology, University of Mississippi Medical Center, Jackson, MS 39216


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
To identify differentially expressed genes from channel catfish macrophages, a cDNA library from LPS-stimulated catfish macrophages was screened by subtractive hybridization. This screening yielded a 552-bp cDNA coding for catfish thioredoxin (CF-TRX). The deduced amino acid sequence revealed that CF-TRX contains 107 amino acids and is 59% homologous to human adult T cell leukemia-derived factor/TRX, originally described as an IL-2R{alpha}-inducing factor. Northern blot analyses showed that CF-TRX is expressed in catfish T and macrophage cell lines, but weakly in B cell lines. Similar results were also observed in Western blot analyses using a mAb specific for recombinant CF-TRX (rTRX). The use of rTRX in functional studies demonstrated that rTRX induces in vitro proliferative responses of catfish PBL that were synergistically enhanced by the addition of culture supernatants from catfish T cell lines. In addition, cell separation studies and flow cytometric analyses revealed that the cells proliferating in rTRX-stimulated cultures were mostly B cells. These results suggest that CF-TRX may have an important role in the activation and proliferation of channel catfish B cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Thioredoxin (TRX)4 was originally isolated from Escherichia coli and characterized as the hydrogen donor for ribonucleotide reductase, an essential enzyme for forming deoxyribonucleotides (1). It is a small, ~12-kDa multifunctional protein with a redox-active disulfide/dithiol within the conserved active site sequence of CGPC. It is believed to be present in all eukaryotic and prokaryotic organisms (2, 3).

In humans, it was first isolated from culture supernatants of an adult T cell leukemia. It was termed adult T cell leukemia-derived factor and found to induce IL-2R{alpha} expression (4, 5). Human TRX cDNA was first cloned from human T cell leukemia virus-I-infected T cells and EBV-infected B cells (6, 7). A single copy of the human TRX gene has been mapped to the short arm of chromosome 3 (8). In mice, a single copy of a functional TRX gene was mapped to chromosome 4, and a TRX pseudogene was located on chromosome 1 (9). In the yeast Saccharomyces cerevisiae, two TRX genes, TRX1 and TRX2, were mapped to chromosomes XII and VII, respectively (10). It has been demonstrated that TRX can be secreted by lymphocytes and other cell types (11, 12, 13) via a nonclassical leaderless pathway (12); detectable levels are found in human serum (14, 15).

Recently, it has been shown that TRX is stress-inducible, with both intracellular and extracellular functions (16). Intracellularly, TRX is involved in the regulation of protein-protein or protein-nucleic acid complexes through the reduction/oxidation of protein cysteine residues. For example, the DNA-binding activities of NF-{kappa}B (17), p53 (18), and the jun/fos complex (19, 20) are enhanced by TRX. Furthermore, TRX is required for DNA binding of the glucocorticoid receptor (21) and for optimal interaction between transferrin receptor mRNA and the iron-responsive element binding protein (22). Extracellularly, TRX synergistically enhances DNA synthesis of proliferating cells in combination with a number of cytokines, such as IL-1, IL-2, IL-4, IL-6, and TNF (23, 24, 25). It also has been shown that human TRX stimulates human fibroblasts (7, 26) and several types of tumor cell lines (27, 28) and significantly prolongs survival of B-type chronic lymphocytic leukemia cells (29). Interestingly, it was found that the expression of murine and human cytokines including IL-1{alpha}, IL-2, IL-6, IL-8, and TNF is strongly up-regulated by TRX (30). Such studies suggest that TRX may play an important costimulatory role in cytokine expression.

The present study reports the initial isolation and characterization of a full-length TRX cDNA clone from a teleost, the channel catfish. In addition, the growth-promoting activity of recombinant catfish TRX (CF-TRX) was investigated. The results demonstrate that TRX is highly expressed in catfish T and macrophage cell lines and appears to have an important role in the activation and proliferation of catfish B cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Experimental animals and PBL isolation

Channel catfish (Ictalurus punctatus), 1–2 kg, were obtained and maintained in individual tanks as described previously (31). Blood was drawn from the caudal vein of tricaine methanesulfonate-anesthetized fish into heparinized Vacutainers, and PBL were isolated by centrifugation over Accu-Prep solution (Nycomed Pharma, Oslo, Norway) as described previously (32).

PBL and cell line cultures

The catfish clonal cell lines used in this study were 1G8 and 3B11 (B cell lines; see Ref. 33), 75C.2, 42TA3.3, and 28S.3 (T cell lines), and 42TA (macrophage cell line); each of these cell lines appears to be immortal, i.e., they grow without the need for restimulation. TS.32.15 is an Ag-specific CTL line that requires weekly stimulation with irradiated 3B11 cells for continued proliferation (34). PBL and cell lines were cultured at 27°C in a humidified 5% CO2 atmosphere in AL medium (AIM-V/L-15; Life Technologies, Rockville, MD) supplemented with 4% heat-inactivated catfish serum (AL-4) as described previously (32).

Mitogen stimulation and culture supernatants

Mitogenic stimulation of catfish PBL was performed as previously described (32). Briefly, LPS from Salmonella typhimurium at a concentration of 500 µg/ml was used to stimulate B cell and macrophage lines. Con A at a concentration of 50 µg/ml was used to stimulate T cell lines. Freshly isolated PBL were pulsed for 12 h with a mixture of PMA and calcium ionophore A23187 at concentrations of 1 µg/ml and 10 µg/ml, respectively; PMA/A23187-containing medium was then replaced with fresh AL-4 medium. All reagents were purchased from Sigma Chemical (St. Louis, MO). Culture supernatants from immortal T (75C.2) and macrophage (42TA) cell lines and PMA/A23187-stimulated PBL were used as sources of putative cytokines in proliferation assays.

Isolation of channel catfish macrophages

Freshly isolated catfish PBL were incubated for 3 h at 107/cm2 in 155-cm2 petri dishes at 27°C. Nonadherent cells were removed, and the adherent cells (macrophages) were washed five times with RPMI 1640 (Sigma Chemical). The adherent cells (macrophages) were cultured in the presence or absence of 100 µg/ml LPS for 8–12 h at 27°C followed by isolation of total RNA.

Molecular cloning of CF-TRX cDNA

Total cDNA was synthesized from 1 µg of poly(A)+ RNA extracted from unstimulated and LPS-stimulated macrophages using the Timesaver cDNA synthesis kit (Amersham Pharmacia Biotech, Piscataway, NJ). The cDNA from LPS-stimulated cells was ligated to Lambda ZAP II using EcoRI linkers (Stratagene, La Jolla, CA). The LPS-stimulated macrophage cDNA library was screened by subtractive hybridization. Briefly, two replicate copies of the cDNA library transformants were blotted on nitrocellulose filters. One set of filters was hybridized with total [32P]cDNA from LPS-stimulated macrophages. The duplicate set of filters was hybridized with total [32P]cDNA synthesized from unstimulated macrophages. Plaques that were differentially expressed and hybridized only to stimulated total [32P]cDNA were isolated and sequenced. Two of the cDNAs sequenced were identical and, as indicated below, encoded for CF-TRX.

RNA isolation and Northern blot analysis

Total RNA was extracted from unstimulated and stimulated PBL, isolated macrophages, and 1G8, 3B11, 42TA3.3, 28S.3, 42TA, and TS.32.15 cell lines according to the manufacturer’s instructions, using RNAzol B solution (Tel-Test, Friendswood, TX). RNAs (5–10 µg) were separated on 1% agarose gels containing formaldehyde and transferred to Hybond-N membranes (Amersham Pharmacia Biotech). The CF-TRX DNA and a catfish 18S rDNA were 32P-labeled by random primers using Megaprime DNA-labeling systems (Amersham Pharmacia Biotech) and served as probes.

Preparation and isolation of CF-rTRX

The open reading frame of CF-TRX cDNA was cloned into the bacterial expression vector pQE-30, expressed in E. coli as a histidine fusion protein and purified on a Ni-nitrilotriacetic acid agarose column according to the manufacturer’s procedures (Qiagen, Valencia, CA). CF-rTRX was aliquoted and stored at -20°C until used. mAb, designated anti-CF-rTRX, was generated using purified CF-rTRX according to standard methods (35). This mAb was an IgA {kappa} protein; the isotype-matched negative control was mouse protein TEPC 15 (Sigma).

Immunodetection of TRX in catfish cell lysates

Cell lines 3B11, 1G8, 28S.3, 42TA, and TS.32.15 were solubilized in 100 µl of lysis buffer (20 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1 mM MgCl2, 1 mM KCl, 50 mM EDTA) containing 1% BSA, 1% Nonidet P-40, 1 mM PMSF, 5 µg/ml aprotinin, 10 µM leupeptin, and 1 mM iodoacetamide. Cell equivalents (2 x 106) from each lysate were electrophoresed on 12% SDS polyacrylamide gels under reducing conditions. After electrotransfer to nitrocellulose Hybond ECL (Amersham Pharmacia Biotech), the membranes were blocked with 5% fat-free milk and incubated with anti-CF-rTRX mAb. The blots were washed, incubated with goat anti-mouse Ig conjugated with HRP, and developed using the ECL Western blot detection kit (Amersham Pharmacia Biotech) according to the manufacturer’s instructions.

In vitro PBL cultures

For flow cytometric and Northern blot analysis, freshly isolated PBL were cultured at 5 x 106/ml in 75 cm2 tissue culture flasks with different doses (0, 1, and 10 µg/ml) of CF-rTRX. The cultures were sampled at days 0, 2, 4, and 7. For proliferation assays, freshly isolated catfish PBL (unfractionated and sorted) were cultured in 96-well culture plates in triplicate at 5 x 105 cells/well in the presence of different doses (0, 0.2, 1, and 5 µg) of CF-rTRX. In addition, culture supernatants (10% of the final volume) from 75C.2 or 42TA cell lines or activated PBL were combined with CF-rTRX. The cells were cultured in AL-4 medium for 3 days at 27°C in humidified 5% CO2-air. Eighteen hours before harvesting, the cells were pulsed with 0.5 µCi [3H]thymidine and subjected to water lysis onto glass fiber filters. The incorporation of [3H]thymidine was measured using a Matrix 96 direct beta counter (Packard Instrument, Downers Grove, IL). Viable cell counts were performed by light microscopy in a hemocytometer using trypan blue exclusion.

mAbs and flow cytometry analysis

The mAbs used in flow cytometric analysis were 1.14, an anti-rainbow trout IgM (36) as the isotype control, and 9E1, an anti-catfish IgM (37, 38). For surface staining, 106 cells in 50 µl RPMI 1640 were incubated on ice for 30 min with 50 µl of culture supernatant containing one of the mAbs mentioned above. The cells were washed with cold RPMI 1640 containing 0.02% NaN3, resuspended in 50 µl RPMI 1640 containing 1 µg of PE-labeled goat anti-mouse Ig (Southern Biotechnology Asociates, Birmingham, AL), and incubated on ice for 10 min. After washing with RPMI 1640-NaN3, the cells were resuspended in 0.5 ml RPMI 1640-NaN3 and analyzed for surface staining by flow cytometry using a BD Biosciences (San Jose, CA) FACScan system.

Sorting B cells from freshly isolated PBL

Catfish B cells were sorted from freshly isolated PBL using MACS goat anti-mouse IgG microbeads (Miltenyi Biotec, Auburn, CA) according to the manufacturer’s instructions, with some modifications. Briefly, 3–4 x 108 PBL were resuspended in 5 ml of culture supernatant containing mAb 9E1 and incubated on ice for 30 min. The cells were washed with cold RPMI 1640, resuspended in 800 µl of RPMI 1640, mixed with 200 µl of MACS goat anti-mouse IgG microbeads, and incubated for another 30 min on ice. The cells were mixed with 1 ml of PE-labeled goat anti-mouse IgG for an additional 5–10 min. Cells were washed and resuspended in 2 ml of cold RPMI 1640 containing 3% FBS. The cell suspension was applied to a LS+/VS+ column (Miltenyi Biotec) that was then placed in a magnetic field; the surface Ig-negative cells (sIg-; non-B cells) passed through the column. The column was washed with 9 ml RPMI 1640–3% FBS and the adherent sIg+ (B cells) were flushed from the column in 5 ml RPMI 1640–3% FBS using a plunger. Samples from unfractionated PBL, and sIg- and sIg+ cells, were analyzed by flow cytometry to determine the purity of the sorting process; it usually ranged from 90 to 95%.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
CF-TRX expression

A cDNA library from LPS-stimulated catfish macrophages was screened by subtractive hybridization, and a 552-bp cDNA coding for CF-TRX was isolated (Fig. 1Go). This cDNA has an open reading frame of 324 bp, starting with the first ATG codon located at nucleotide 32 and ending with a TGA termination codon. It codes for 107 amino acids, with an estimated molecular mass of 11,951 Da and an isoelectric point (pI) of 5.51. There are no potential N-linked glycosylation sites. An amino acid alignment comparing human, mouse, chicken, yeast (Coprinus comatus), and E. coli TRXs to CF-TRX is shown in Fig. 2Go. These molecules show 59, 57, 55, 44, and 21% identity to CF-TRX, respectively. Each contains the highly conserved redox-active disulfide/dithiol site, CGPC.



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  		FIGURE 1. Nucleotide and inferred amino acid sequences of the CF-TRX cDNA clone. The inferred amino acids (single letter code) are shown above the second base of each codon, and the number of nucleotides and amino acids are at the right margin. The conserved disulfide/dithiol site, CGPC, is boxed. The termination codon is indicated by an asterisk (*).

 


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  		FIGURE 2. Alignment of different TRX protein sequences. The amino acid residues are numbered on the right. The different sequences are compared with CF-TRX. Dots indicate identical amino acids and gaps (-) are inserted to optimize the alignment. The conserved active site (CGPC) is indicated in boldface letters. Accession numbers are: catfish, AF293651; human, p10599; mouse, p10639; chicken, p08629; C. comatus, cab52130.1; E. coli, p00274.

 
Expression of TRX in catfish PBL and cell lines

Northern blot analyses of RNA extracted from stimulated and unstimulated PBL and several types of cell lines were performed with 32P-labeled CF-TRX cDNA (Fig. 3Go, upper panel). The expression of CF-TRX (0.6-kb transcript) varied among different cells. It was expressed at moderate to high levels in T cell lines (28S.3 and TS.32.15) and at somewhat lower levels in the macrophage cell line (42TA) and PBL. In contrast, TRX expression was detected at very low levels in B cell lines (3B11 and 1G8). Stimulation of cell lines and PBL with various mitogens appeared to have little or no effect on TRX expression. Control hybridization performed using catfish 18S rDNA as a probe showed similar RNA loads for all samples (Fig. 3Go, lower panel).



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  		FIGURE 3. Northern blot analyses showing expression of TRX in catfish PBL and various leukocyte cell lines. Total RNA was isolated from unstimulated and stimulated (stim.) clonal B (3B11, 1G8), T (42TA3.3, 28S.3), macrophage (42TA) and cytotoxic T (TS.32.15) cell lines as well as freshly isolated PBL. RNA (10 µg/lane) was separated on a 1% agarose formaldehyde gel and transferred to a nylon membrane. Blots were hybridized with 32P-labeled catfish TRX probe and an 18S rDNA probe as a control panel. The results are representative of three independent Northern blot analyses.

 
Cell lysates from various catfish cell lines were subjected to SDS-PAGE under reducing conditions, and immunodetection was performed using anti-CF-rTRX mAb (Fig. 4Go). Western blot analysis detected a single protein with an apparent molecular mass of ~12 kDa. Protein was present at high levels in the 28S.3 and TS.32.15 cell lines; and lower levels were detected in the 42TA, 1G8, and 3B11 cell lines. Purified CF-rTRX was used as a positive control (Fig. 4Go, lane 1).



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  		FIGURE 4. Western blot analysis demonstrates TRX protein expression by catfish leukocyte lines. Cell lysates (2 x 106 cell equivalents) from B (1G8, 3B11), T (28S.3), macrophage (42TA), and cytotoxic T (TS.32.15) cell lines were subjected to 12% SDS-PAGE and transferred to a nitrocellulose membrane. CF-rTRX was loaded in the first lane as a positive control. Membranes were incubated with anti-CF-rTRX, washed, incubated with goat anti-mouse Ig conjugated to HRP, and developed by ECL. The results are representative of three independent Western blot analyses.

 
Growth-promoting effect of CF-rTRX

To test the effect of CF-rTRX on PBL proliferation, the recombinant fusion protein was used either alone or in the presence of T cell (75C.2), macrophage (42TA), or stimulated (PMA/calcium ionophore A23187) PBL culture supernatants (Fig. 5Go). CF-rTRX significantly promoted PBL proliferation in a dose-dependent fashion. The combination of CF-rTRX and various leukocyte culture supernatants each synergistically enhanced the incorporation of [3H]thymidine in catfish PBL. To better assess proliferation, cell counts were taken after 3 days. These counts revealed an ~2.5-fold increase (over day 0) in total cells present in the CF-rTRX-treated cultures, and a ~3.5-fold decrease in total cells in the control cultures. These data clearly support the argument that CF-rTRX induces proliferative responses in catfish PBL.



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  		FIGURE 5. CF-rTRX enhances the proliferation of freshly isolated PBL. Catfish PBL (5 x 105) were cultured for 3 days at 27°C with 0, 0.2, 1, or 5 µg/well CF-rTRX in the absence or presence of culture supernatants (10%) from T cell line 75C.2, macrophage cell line 42TA, or PMA/calcium ionophore A23187-stimulated PBL. Eighteen hours before harvesting by water lysis, the cells were pulsed with 0.5 µCi of [3H]thymidine. The results are representative of six different fish.

 
To determine which lymphocyte types are affected by CF-rTRX, catfish PBL were cultured with CF-rTRX (1 and 10 µg/ml), and samples were collected after 0, 2, 4, and 7 days in culture. Flow cytometric analysis of the cell samples using mAb 9E1 revealed that the addition of CF-rTRX to PBL cultures induced increases in the percentage of B cells (Fig. 6GoA). After 7 days, 44 and 65% of cells in cultures treated with 1 and 10 µg/ml CF-rTRX, respectively, were B cells. In contrast, the percentage of B cells in control cultures (untreated with CF-rTRX) decreased to 9% after 7 days. Northern blot analysis using a catfish Cµ1 probe was performed on RNA extracted from the same cultures and revealed that expression of IgM mRNA was up-regulated in the CF-rTRX-treated cultures (Fig. 6GoB, upper panel). In control cultures, the µ transcript was detected at very low levels at all time points. Control hybridization using catfish 18S rDNA as a probe showed similar levels of RNA in all samples (Fig. 6GoB, lower panel). Each of the above observations supports the notion that B cell numbers and µ-chain expression are increased in CF-rTRX-treated cultures.



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  		FIGURE 6. Catfish B cells are increased in culture by the stimulation of PBL with CF-rTRX. PBL were cultured with 0, 1, and 10 µg/ml CF-rTRX, and cell samples were collected after 0, 2, 4, and 7 days in culture. A, Each sample was assayed for surface IgM with mAb 9E1 (specific for catfish µ-chain) by flow cytometry using a FACScan system. Anti-trout IgM mAb 1.14 was used as an isotype control. B, Total RNA was isolated from each sample and assayed for µ expression by Northern blot analysis. The RNA (5 µg) from each sample was separated on a 1% agarose formaldehyde gel and transferred to a nylon membrane. Blots were hybridized with 32P-labeled catfish Cµ1 exon probe and 18S rDNA. The results are representative of three independent experiments.

 
To demonstrate that CF-rTRX exerts a direct effect on B cells, freshly isolated PBL were magnetically sorted into sIg+ (B cells) and sIg- (non-B cells) using mAb 9E1 and cultured in the presence of CF-rTRX. As shown in Fig. 7GoA, CF-rTRX promoted the proliferation of unfractionated PBL, but appeared to have no effect on sIg- cells. The proliferative effect of CF-rTRX was most pronounced with the sIg+ B cells, especially at 5 µg/well. CF-rTRX-treated sIg+ cell cultures showed an ~3-fold increase in cell numbers after 3 days. In contrast, control sIg+ cell cultures showed an ~10-fold decrease in cell numbers. This proliferative effect was synergistically enhanced when the sIg+ B cells were cultured in the presence of culture supernatants from a T cell line (75C.2), a macrophage cell line (42TA), or activated PBL (Fig. 7GoB). In contrast, the combination of CF-rTRX and culture supernatants did not synergistically enhance proliferation of sIg- cells above the level observed with culture supernatant alone (data not shown). This stimulatory effect of CF-rTRX on the proliferation of sIg+ B cells can almost be completely abolished by adding anti-CF-rTRX mAb to the culture (Fig. 8Go). This inhibition of CF-rTRX-induced proliferative responses is more effective at the lower doses (0.2 and 1 µg) of CF-rTRX; the isotype-matched control did not affect the proliferative responses of cells treated with CF-rTRX (data not shown). To determine whether the CF-rTRX growth-promoting effect was caused by possible LPS contamination of the CF-rTRX preparation, sIg+ B cells were cultured with LPS in the presence of mAb against CF-rTRX (Fig. 8Go). The growth-promoting response of LPS on sIg+ B cells was not affected by the addition of mAb against CF-rTRX to the assay, indicating that the proliferative effect of CF-rTRX was specific. In addition, catfish recombinant {beta}2-microglobulin, similar in m.w. to CF-rTRX and prepared in exactly the same way, had no effect on the proliferation of sIg+ B cells (data not shown). The synergistic effect on the proliferation of CF-rTRX-treated sIg+ B cells resulting from use of the above culture supernatants was abolished when mAb against CF-rTRX was added to the cultures (Fig. 9Go). These results support the notion that CF-rTRX has a specific effect on the activation and proliferation of B cells.



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  		FIGURE 7. CF-rTRX has a direct effect on catfish B cell proliferation. Freshly isolated catfish PBL were separated into sIg+ and sIg- populations using mAb 9E1 by MACS. A, B cells (sIg+) and non-B cells (sIg-) as well as unfractionated cells were cultured for 3 days at 27°C with 0, 0.2, 1, or 5 µg/well of CF-rTRX. B, B cells (sIg+) were cultured for 3 days at 27°C with 0, 0.2, 1, or 5 µg/well of rTRX in the presence or absence of culture supernatant from T cell line 75C.2, macrophage line 42TA, or PMA/calcium ionophore A23187-stimulated PBL. Eighteen hours before harvesting, the cells were pulsed with [3H]thymidine. The results are representative of five independent experiments.

 


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  		FIGURE 8. Anti-CF-rTRX mAb blocks the in vitro B cell growth-promoting activity of CF-rTRX. Freshly isolated B cells (sIg+) (5 x 105) were cultured for 3 days at 27°C with 0, 0.2, 1, or 5 µg/well of CF-rTRX or 20 µg/ml LPS in the presence or absence of 5 µg of anti-CF-rTRX mAb. Eighteen hours before harvesting, the cells were pulsed with [3H]thymidine. The results are representative of four independent experiments.

 


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  		FIGURE 9. Anti-CF-rTRX mAb blocks the synergistic growth-promoting activity of culture supernatants. Freshly isolated B cells (sIg+) (5 x 105) were cultured for 3 days at 27°C with 0, 0.2, 1, or 5 µg/well of CF-rTRX in combination with culture supernatants from T cell line 75C.2, macrophage line 42TA, or PMA/calcium ionophore A23187-stimulated PBL in the presence or absence of 5 µg of anti-CF-rTRX mAb. Eighteen hours before harvesting, the cells were pulsed with [3H]thymidine. The results are representative of three independent experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Previous studies have shown that growth-promoting factors are produced by channel catfish lymphocytes. For example, catfish LPS-stimulated monocytes produce IL-1-like factors that enhance the proliferation of monocyte-depleted PBL (39). Moreover, culture supernatants from catfish T cell lines contain factor(s), presumably IL-2, with growth-promoting activity for catfish PBL (40, 41). None of these factors have been purified or well characterized.

In attempting to clone and characterize differentially expressed factors/proteins involved in the immune response of catfish, a cDNA clone coding for TRX was identified. The inferred amino acids of CF-TRX showed high homology with vertebrate (human, mouse, and chicken) TRX. In contrast, lower homologies with C. comatus and E. coli TRX were observed. However, the active site motif of CGPC showed absolute identity between each TRX examined. In mammals, especially humans, TRX is well characterized and is considered to have a wide spectrum of functions affecting the immune system and cell activation. In fish, TRX was partially sequenced in the Japanese flounder using the expressed sequence tags technique (42), but no functional studies were performed. The study reported here describes not only the cloning and isolation but also the possible immune function of TRX in channel catfish. Comparison of the deduced amino acid sequences of catfish and flounder TRX (accession number AU050717) showed 70% similarity (data not shown). In contrast, human and mouse TRX share 88% similarity. These data indicate that the diversity between fish may be much higher than in other taxa. The calculated pI of CF-TRX (5.51) is higher than that of the TRX of humans, mice, chickens, yeast, and E. coli, which have calculated pI values ranging from 4.51 to 5.01. It is not known whether these differences in pI values may affect the function or the activation pathway of CF-TRX.

In the present study, CF-TRX expression and function appear to have diverged from TRX of other species, especially humans. For example, CF-TRX was expressed in diploid nontransformed long-term T cell lines and in unstimulated freshly isolated PBL. In contrast, human TRX is highly expressed in virally transformed lymphoid lines, mitogen-stimulated cell lines, or stimulated PBL, and expressed poorly, if at all, in nontransformed or unstimulated cells (6, 7, 23). In contrast to the situation with human cells, stimulation of catfish cell lines or PBL with mitogens did not affect the expression of TRX. The fact that CF-TRX is expressed in freshly isolated and unstimulated PBL suggests that it is not a stress-inducible protein as in humans (16). TRX in catfish appears to have an important role within the immune system because it is constitutively expressed in PBL. CF-rTRX promoted proliferation of PBL that was synergistically enhanced by the use of culture supernatant, presumably containing growth-promoting factors from catfish T and macrophage cell lines, and activated PBL. This is similar to the situation in humans, where the combination of rTRX with IL-1, IL-2, IL-4, IL-6, or TNF synergistically enhances proliferation of several types of lymphoid cell lines and PBL (23, 24, 25, 43).

An impressive and thus far unique direct effect of CF-rTRX involved the activation and proliferation of freshly isolated B cells from normal catfish. Flow cytometry revealed that B cells predominated (up to 65%) in CF-rTRX-treated cultures of catfish PBL. Furthermore, Northern blot analysis revealed that the expression of IgM mRNA was highly up-regulated in such treated cultures. In humans, the combination of human rTRX with different types of cytokines enhanced the proliferation of EBV-transformed (3B6) and leukemic (B-CLL) B cells (23, 25, 43). In addition, normal tonsillar B cells that were frozen and subsequently preactivated for 2 days with Staphylococcus aureus Cowan I particles followed by treatment with rTRX showed an effect similar to, albeit lower than, that of the B-CLL cells. However, rTRX used alone had little effect on human B cells (25), in contrast to the situation observed here with catfish B cells.

Because it is known that TRX strongly up-regulates the expression of several cytokines (30) in both humans and mice, it was necessary to determine whether CF-rTRX activates catfish B cells directly or indirectly through an effect(s) on other cell types present in the culture. The cell separation data suggest that CF-rTRX directly promotes the growth of isolated catfish B cells without pretreatment, preactivation, or the addition of exogenous factors. Moreover, this effect was synergistically enhanced in the presence of culture supernatants, presumably containing as yet unidentified cytokine-like factors derived from activated catfish lymphoid cells. It seems that CF-rTRX has a specific direct effect on the activation and proliferation of catfish B cells.

Because CF-rTRX was expressed and purified from an E. coli expression system, it was important to rule out the possibility that the effects observed were due to contamination of the recombinant protein with bacterial LPS. To this end, two experiments that ruled out LPS as a possible contributory factor were conducted. First, purified anti-CF-rTRX mAb, but not an isotype control, almost totally abolished the proliferative effect of CF-rTRX, but not that of LPS, on B cells. Second, catfish recombinant {beta}2-microglobulin (44), having a similar m.w. and produced using the same expression system, was found to have no effect on the proliferation of isolated catfish B cells. When taken together, these results suggest that the purified CF-rTRX used in this study was not contaminated with bacterial LPS, and its growth-promoting effect on B cells was both specific and direct. Additional evidence for the specificity of CF-rTRX was achieved when the synergistic effect of culture supernatants on B cell proliferation was eliminated by the addition of anti-CF-rTRX mAb to the cultures. Still, there is no formal proof that the culture supernatants used in this study contained homologues to mammalian cytokines or IL-like factors. However, results presented here and in previous studies strongly indicate that catfish-activated PBL and several leukocyte lines produce cytokine-like factors.

In conclusion, the present study demonstrates that CF-TRX not only shares some properties with mammalian TRX, but also may have a unique function as a B cell growth factor in teleosts. Work is in progress to clarify the in vivo role of TRX in catfish and to develop a sensitive ELISA to allow the detection of secreted CF-TRX in serum and cell culture supernatants. Studies are also planned to determine the intracellular activation pathway of CF-TRX on fish B cells. The unique ability to culture catfish leukocytes and develop functionally active lymphocyte lines should enable in vitro studies designed to determine the functional role and intracellular signal transduction pathways used by catfish TRX to activate B cells.


   Footnotes
 
1 This work was supported by grants from the National Institutes of Health (AI-19530) and the U.S. Department of Agriculture (95-37204-2225). Back

2 The sequence presented in this article has been submitted to GenBank under accession number AF293651. Back

3 Address correspondence and reprint requests to Dr. L. William Clem, Department of Microbiology, University of Mississippi Medical Center, 2500 North State Street, Jackson, MS 39216-4505. Back

4 Abbreviations used in this paper: TRX, thioredoxin; CF-TRX, catfish TRX; AL medium, AIM-V/L-15 medium; AL-4, AL medium supplemented with 4% heat-inactivated catfish serum; pI, isoelectric point; SIg-, surface Ig-negative cells. Back

Received for publication September 25, 2000. Accepted for publication December 12, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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