Transforming Growth Factor Beta (TGFβ) Is Produced by and Influences the Proliferative Response of Xenopus laevis Lymphocytes

Both TGF/β2 and 5 have been described in the South African clawed frog Xenopus laevis and have been cloned from the tadpole-derived fibroblast cell line, XTC. Because TGFβ has such a profound inhibitory effect on the mammalian immune system, this study was performed to determine whether TGFβ: (a) has any in vitro effects on the growth of Xenopus lymphoblasts, and (b) is produced by mitogen-activated Xenopus lymphocytes. Following stimulation with mitogen or alloantigen, T lymphocytes from Xenopus secrete a T-cell growth factor (TCGF) that is functionally homologous to mammalian interleukin-2 (IL-2). Both recombinant human TGFβ1 and Xenopus TGFβ5 inhibit TCGF-induced proliferation of Xenopus splenic blasts and this inhibition can be reversed with anti-pan TGFβ antiserum. The Xenopus mitogen-induced saturated ammonium sulfate precipitated TCGF-containing supernatant (SAS TCGF SN) also contains latent TGFβ as assayed on mink lung fibroblasts and Xenopus splenic blasts, and experiments utilizing anti-TGFβ antiserum showed that only TGFβ5 is present in this supernatant.


INTRODUCTION
TGFfl is a pleiotrophic cytokine produced by a number of different cell types including platelets, macrophages, fibroblasts, and T and B lymphocytes. TGFfl is secreted in a 100-kD biologically inactive latent form. This latency peptide can be cleaved by a change in pH or proteolysis to yield a 25-kD disulfide-bonded homodimer (reviewed in Roberts and Sporn, 1990). Five types of TGF/J have been cloned: TGFfll, 2, and 3 were originally described in humans (Assoian et al., 1983;Wrann et al., 1987;ten Dikje et al., 1988); TGF/J4 has been found only in chickens (Jakowlew et al., 1988); TGF//5 has been found only in Xenopus (Roberts and Sporn, 1990). The amino-acidsequence identity of the aforementioned processed TGF]/s (after cleavage of latency peptide) is between 60% and 80%. TGF//5 is 76% identical to TGFfll, 66% identical to TGFfl2, 69% identical to TGF]3, and 72% identical to TGFfl4. Regions *Corresponding author. of identity include a highly conserved site and nine conserved cleavage cysteine residues. TGFfl 1 through 5 also show functional conservation in a number of assays, including the inhibition of proliferation of mink lung fibroblasts (MLF) and the stimulation of normal rat kidney (NRK) fibroblast colony formation in the presence of epidermal growth factor (Roberts and Sporn, 1990).
TGF//plays a role in the control of the formation of extracellular matrices, myogenesis, formation and remodeling of bone, and in embryogenesis. TGF// also functions in the immune system. It inhibits Tand B-cell proliferation, NKcell activity, and generation of mixed lymphocyte responses and cytotoxic T cells (Massague, 1990). Kehrl et al. (1986) (Roberts and Sporn, 1990). Because TGFfl has such a profound effect on the mammalian immune system, this study was performed to determine whether TGFfl has any in vitro effects on the growth of Xenopus lymphoblasts and if it is produced by mitogen-activated Xenopus lymphocytes.

RESULTS
Xenopus SAS TCGF SN contains a TCGF that is functionally homologous to mammalian interleukin-2 (IL-2)  Haynes and Cohen, 1993). One of its main in vitro activities is the induction of proliferation of activated, but not resting, splenocytes. Recombinant Xenopus TGFfl5 (rTGFfl5) inhibited the SAS TCGF SN-induced proliferation of 3-day-old Xenopus splenic blasts (Fig. 1A). Maximum inhibition was seen with 0.125 ng/ml and this inhibition could be titrated until no inhibition was seen with 0.001 ng/ml. Similar results were obtained with recombinant human TGFfll (Fig. 1B).
To determine whether the inhibition of lymphoblast proliferation associated with rTGFfl5 resulted from TGFfl5 or from some contaminant  in the TGF5 preparation, TGFfl5 was preincubated with anti-pan TGFfl antiserum and then assayed ( Fig. 2). Whereas the anti-pan TGFfl antiserum reversed the inhibition of proliferation, the control.anti-TGFfl2 antiserum (which does not recognize TGFfl5) did not. The observed inhibition of proliferation, therefore, was specifically due to TGFfl5. Neither antibody was mitogenic for Xenopus blasts (Fig. 2 (Fig. 5). Finally, because both TGFfl2 and 5 have been described in Xenopus (Roberts and Sporn, 1990), antiserum specific for each of these proteins was used in an attempt to neutralize the TGFfl biological activity found in the acid-treated SAS TGCF SN. Figure 6 shows that the anti-TGFfl2 has no effect, whereas the anti-TGFfl5 reverses the inhibitory activity found in the acid-treated supernatant.

DISCUSSION
In mammals, TGFfl is produced by mitogen-activated T cells, inhibits IL-2-dependent T-cell proliferation, and downregulates the immune response (Kehrl et al., 1986). Our study shows that these observations also are applicable to the antiserum treatment. Whether "all" supernatants prepared from mitogen-or antigen-stimulated larval as well as adult Xenopus splenocytes also contain only TGFfl5 is currently being investigated. Because TGFfl is involved in development, we are also investigating its role in the immune system during metamorphosis to determine if the downregulation of the immune response that is seen during this period (Flajnik et al., 1987) Pasquier, 1990). Xenopus leukocytes produce an IL-l-like cytokine . Xenopus T cells produce additional cytokines involved in Tand B-cell proliferation (Cohen and Haynes, 1991) and also exhibit MHC-restricted cytotoxicity (Harding, 1990 Goat IgG FIGURE 6. Anti-TGFfl5, but not anti-TGFfl2, antiserum reverses TGFfl inhibitory activity found in SAS TCGF SN. Untreated or acid Rx SAS SN (25% per well) with or without goat anti-TGFfl2, goat anti-TGFfl5, or control goat IgG, at the indicated concentrations, was assayed (as described in Materials and Methods) on 3-day-old Xenopus splenic blasts in a 3-day 3H-thymidine incorporation assay. Results are expressed as mean CPM+SE. CPM with medium alone is 2061.5+80.1. mammalian IgM, IgG, and IgA (Du Pasquier, 1989). By demonstrating that Xenopus lymphocytes produce TGFfl that can downregulate lymphocyte proliferation, the present study provides additional evidence for the remarkable similarity of the Xenopus and mammalian immune systems.

Animals
Fully grown adult female Xenopus laevis were purchased from Xenopus 1 (Ann Arbor, Michigan) or the South African Xenopus Facility (Noordhoek, South Africa).

Production of SAS TCGF SN
SAS TCGF SN was generated as previously described  Xenopus TGF//5 was purchased from R & D Systems) or the supernatants to be tested were added to the cultures that were then incubated for 24 hr. Each well was then pulsed with 1/Ci 3H-thymidine (Amersham) for 6 hr and freeze/thawed before harvesting and processing for liquid scintillation spectrometry. All assays were plated in triplicate and the data are presented as the mean CPM+SE.