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Differentiation of B cells in the bovine. Role of cytokines in immunoglobulin isotype expression
Veterinary
Veterinary
ELSEVIER
Immunology and Immunopathology 54(1996) 61-67
immunology and immunopathology
Differentiation of B cells in the bovine. Role of cytokines in immunoglobulin isotype expression D. Mark Estes University oj’Missouri,
College
of Veterinwy
Medicine, Depurtment
ofVeterinary
Pathohiology.
Columhiu,
MO 65211. USA
Abstract
Cytokines such as IFN-y, IL-4 and TGF-/3, are known to regulate Ig class switching by positively or negatively regulating germline C, transcription in the mouse and human. Here we show at the protein level that similar regulatory mechanisms are in place in the bovine using various modes of B cell activation and cytokine combinations. Using antigen-receptor cross-linkage or mitogen (pokeweed mitogen) costimulation, we demonstrate that recombinant bovine IL-4 upregulates the expression of IgM, IgG, and IgE in vitro. Upregulation of IgG, and IgE production by rboIL-4 is inhibited in a dose-dependent manner by the addition or rboIFN-y to the cultures. Using similar methodologies, we have also investigated the effects of TGB-P on IgA production in the presence or absence of various cofactors including IL- 10 and IL-2. We find that TGF-/3 will not solely upregulate IgA production from noncommitted precursors (sIgM+) in coculture with mitogen or with forrnalin fixed Staphylococcus cowan Strain I (SAC), but does have positive effects on IgA production in the presence of both IL-2 and IL-IO. These observations suggest that different modes of T-independent costimulation using classical B cell activators does not result in differential IgG subisotype or IgE production by bovine B cells in the presence of cytokines. Together these results suggest that individual cytokines regulate isotype expression in cattle. Keywords:
B cells; Bovine; Cytokines;
Immunoglobin;
Isotope
1. Introduction The bovine immune system differs from other mammalian species with regard to a variety of immune parameters including development of the B cell lineage and the humoral response. For example, cattle appear to lack sIgD and have a unique distribution of immunoglobulin (Ig) isotypes in secretions such as colostrum (Butler, 1983). Due 0165-2427/96/$15.00 PII
Copyright
SO1 65-2427(96)05684-X
0 1996 Elsevier Science B.V. All rights reserved
to fundamental differences in mlmune physiology. cytokine control of antibody production in cattle may differ from the regulatory pathways which have been described in the mouse and human. Recent developments in cell culture systems and the availability of recombinant cytokines have allowed the initial investigations into T cell-B cell interactions. modes of B cell activation/differentiation, and the cytokine regulatory pathways involved in Ig class switching in cattle to begin. Immunoglobulin isotype or class switchin g is the process by which a B lymphocyte can alter expression in Ig heavy chain isotype and express the same VDJ region. altering the effector function of the antibody to a given antigen and thereby expanding the capabilities of the humoral immune response. Class switching in the mammalian species examined to date has been shown to be largely regulated by T cell-derived cytokines and by direct and indirect cognate T cell-B cell interactions (Noelle and Snow. 1990). Studies of T cell-B cell interactions and costimulation have largely focused on the role of the CD40 molecule present on B cells and its counter-receptor on T lymphocytes, the CD40 ligand. These studies have demonstrated that CD40 cross-linking represents the first step in the interaction between T and B lymphocytes and this interaction has profound effects on B cell activation and isotype production (Banchereau et al.. 1994). 7 cell-independent systems of Ig isotype production are suitable for the initial characterization of cytokine regulatory pathways. Cytokines alone are insufficient to effect class switching bv resting B cells and must act in ;I c.ostimulatory or additive fashion with polyclonal B cell activators. For induction of non-IgM isotypes (IgG,, IgG,. IgA. and IgE) in vitro in the bovine. pokeweed mitogen (PWMI with IL-2. SAC, and sheep anti-bovine IgM have been used to support Ig production from resting sIgM’ B cells (Estes et al., 1994; Estes et al.. 1995: Estes et al.. 1996).
2. IFN-7
stimulates
IgG, production
in vitro
Various T-independent costimulation systems have been developed for cytokine costimulation of Ig class switchin g in murine systems in vitro including bacterial lipopolysaccharide (LPSI. polyclonal anti-mouse IgM coupled to agarose beads and anti-IgD dextran (Purkerson and Isakson. 1992; Snapper and Mond. 1993). In the bovine. polyclonal sheep anti-bovine IgM coupled to beads through an avidin-biotin linkage has proven effective in stimulating IgM and non-IgM antibody production on in vitro coculture with crude cytokine containin g culture supernatants or recombinant cytokines (Estes et al.. 1994). Similar results have been reported for PWM with a requirement for IL-2 identified for IgM production in the absence of T cells (Estes et al., 1994; Collins and Oldham. 1995). Resting B cells activated by PWM or sheep anti-bovine IgM coated agarose beads proliferate and upon addition of IFN-y containing supernatants or recombinant bovine IFN-y. secrete mostly IgM and IgG, with no increase in IgG, or IgA levels (Estes et al., 1994). Coculture with recombinant bovine IL-2 potentiated the response by increasing quantitative amounts of Ig produced without qualitatively altering the isotype distribution. Therefore, a predominant T helper cell Type 1 (T,, 1) response in vivo would be implicated in upregulation of IgG, production. In the murine system. T,, I cells have been shown to provide T cell help for IgG?,
D.M. Estes/
Veterinary Immunol~~gy and Immunopathology
54 (1996) 61-67
63
l-
10%
Fig. I. In situ hybridization analysis of anti-p medium alone or 10% T cell culture supematants representative of the percentage of Ig C, region obtained from triplicate cultures for each variable
TCS
stimulated B cells after 48 h reculture in the presence of containing IFN-y in the absence of IL-4 (TCS). Results are mRNA+ cells per 100 total cells as indicated. Cells were and pooled for analysis.
production in vitro (Mosmann and Coffman, 1989). At the transcriptional level, in situ hybridization analysis allows the frequencies of cells producing Ig heavy chain gene segment mRNA for IgG,, IgG, and IgM to be determined on a per cell basis. Analysis of sheep anti-bovine IgM stimulated bovine B lymphocytes at 48 h post-stimulation with IFN-y-containing culture supematants indicated increased frequencies of IgG, mRNA+ cells and reduced frequencies of IgM and IgG, mRNA+ cells relative to control supematants lacking IFN-7 (Fig. 1). These results suggest that IFN-7 controls IgG, production at the transcriptional level.
3. Role of IL-4 in IgG, and IgE production
in cattle
IL-4 and IFN-y in murine systems reciprocally regulate isotype production directly via their actions on B cells and indirectly through T helper cell differentiation pathways leading to T, 1 or T, 2-type responses. In both the mouse and human, IL-4 is integrally involved in the regulation of IgE synthesis and this process can be directed through intermediate switching to IgG, and IgG,, respectively (Vercelli and Geha, 1993). In particular, IgE production was absent in mice rendered deficient for IL-4 production by gene targeting via homologous recombination (Kuhn et al., 1991). In vitro systems which utilize T cells or T cell membranes are complex, therefore we opted to utilize the classical B cell activators (anti-IgM antibodies, SAC, and PWM) to analyze the effects of recombinant bovine IL-4 on Ig isotype production. In these cultures, both PWM and SAC in the presence of rboIL-4 stimulated IgE production in a concentration-dependent manner in vitro from sIgM+ B cells (Fig. 2). These results differ from those obtained with human B cells in which classical activators do not act in concert with IL-4 to stimulate IgE production (Vercelli et al., 1989), again reinforcing the notion that species specificity is an important issue in cytokine regulatory pathways. Consistent with observations in the mouse and human, IL-4 addition to stimulated bovine B cells also resulted in enhanced IgG subclass (IgG,) production (Fig. 2). IgG, production was
64 500
400 T & r300.I e i: 200t I c” TOO.-
N
0
I
%IL~
supernatant added (v/v)
5
v
10
’
20
500
(v/v)
-^ E & 20 c in ‘a 15 r” e 610 w 9
,E
Lm 400 .f E ci
added
25
=‘
2
%IL4 supernatant
300
200
6 c” 100
5
0 %IL~
0 5
10
20
supernatant added (v/v)
Fioc. 2. Fractionated IL--l containing COS cell wpemalanrb uprrgulate cxprcssion of IgM. IpG,. not IgG,
or IgA. (A)
Anti-IgM: SAC + IL2 ml
’ ). IgA
IgM levels (ng ml
’)
in 6 day culture supernalants
( I p*gml ’ ). A, SAC (O.OOS% final concentration); 0. ( I15 II ml ’ ); n (b) IgG, levels (ng ml-- ’ ): CC’) IgG, levels
0,
PWM
and IgE but
as determined by ELISA; PWM + IL2 (ng ml-
’ );
(I 25 U (D)
ml
0,
’ ): A,
IpE levels (ng
levels were equal to medium only cultures under all parameters tested (data not shown). Results
are presented as the mean of triplicate cultures and are representative of three separate experiments from the same donor animal. Standard error values did not exceed 10% of the mean.
induced at relatively lower concentrations than those required for IgE as has been observed in the mouse (Snapper and Mond, 1903). Addition of recombinant bovine IL-2 as a cofactor enhanced synthesis of IgG, and IgE relative to cultures receiving IL-4 only at the same concentrations (Fig. 2). In contrast to LPS-induced murine B lymphocyte cultures receiving IL-4 (Snapper and Paul, 1987). secretory IgM synthesis was also enhanced in a concentration-dependent manner. IgG, and IgE production induced by rboIL-4 were inhibited in a close-dependent manner by IFN-y (Table 11. To further investigate the in vitro activities of IL-4 in the bovine B lineage. the expression of markers known to be affected by IL-4 were also studied in the bovine at 24-48 h following costimulation with classical activators and IL-4 or with IL-4 alone. These markers included IL-2 receptor a-chain (IL-2R). the low affinity IgE receptor CD23, and Class II MHC expression. Flow cytometric analysis of cells cultured for 24 h in the presence of COS-cell derived rboIL-4 alone indicated enhanced expression of all three markers relative to cells cultured in the presence of mock transfected COS cell
D.M. Es!es/
Table 1 Inhibitory
SAC
Immunology
and Immunopathology
effects of IFNy on IgM, IgG,, and IgE secretion
Costimulator Anti-IgM
Veterinary
b
by anti-IgM
Cytokine added
IgM (ng ml- ‘I
0% (v/v) IL-4 20% (v/v) IL-4 20% IL-4+IFNy (100 ng ml-‘) 20%IL-4+IFNy(10ngmll’) 0% (v/v) IL-4 20% (v/v) IL-4 20% IL-4+IFNy (1OOng ml-‘) 20% IL-4+IFNy (lOngml_‘)
llOk9 < 25 43 f 4 < 25 113k12 58+7 82+4
< 25
54 (1996) 61-67
or SAC-stimulated
65
sIgM+
B cells a
IgG, (ng ml- ‘1
IgE (ELISA units) c
18Ok 17 362 f 47 123f8 98k7 125+24 256k51 106+ 19 119f8
< 25 438t4 < 25 < 25 < 25 248 + 33 < 25 < 25
a sIgM+ B cells were obtained from peripheral blood and cultured at 1 X lo5 cells for 6 days and supematants harvested for determination of secreted immunoglobulin levels by ELISA as described in Materials and methods.b biotinylated sheep anti-bovine IgM was added at 1 pg 10e6 cells with avidin coated beads at a 3: 1 bead per cell ratio; SAC was added at 0.005% (v/v) final concentration.c ELISA units ate determined by linear regression relative to a bovine serum standard; data are presented as the mean of triplicate cultures + SEM.
MHC class II
lL2R
CD23
20%114
IL
Frequency
l-
1O%lL4
5%IL4
L
Fluorescence Intensity
c
Fig. 3. Expression of surface IgM, MHC Class II, and IL2R on bovine B cells after 24 h culture with size-fractionated (30 kD > MW > 10 kD) bovine IL-4 from COS cell supematants. Lower panels from bottom to top indicate results obtained by increasing concentrations of IL-4 (O-20%, v/v) in cultures without additional costimulators. Cells were stained with primary antibodies as described in Materials and Methods with mouse anti-bovine MHC Class II (114-B). mouse anti-bovine IL2 receptor (108-A. IL2R). mouse anti-human CD23 (IOB8), murine isotype controls, or biotinylated sheep anti-bovine IgM (The Binding Site) at 1 pg 1O-6 cells. Mouse monoclonal antibodies were detected using biotinylated sheep anti-mouse Ig which had been previously absorbed with a bovine serum agarose column and streptavidin-FITC. Biotinylated sheep primary antibodies were detected using streptavidin-FITC only.
supernatants (Fig. 3). Together these data suggest a broad conservation of the varied activities of IL-4 described for B cells of both mouse and human origin with those in cattle.
4. Regulation
of IgA expression
in the bovine
Transforming growth factor beta (TGF-/?I ) has been established as a switch factor for IgA due to its ability to initiate germline transcripts for the Ig-cu C,, region in the mouse (Coffman et al.. 1989: Sonoda et al.. 19891. TGF-/3 also appears to regulate IgA production in humans (van Vlasselaer et al.. 1992). LPS and PWM were utilized as costimulators for mouse and human B cells in the initial studies of the effects of TGF-,!3, respectively. More recent studies with human B lymphocytes indicate that alternative factors also appear to regulate IgA production utilizing anti-CD40 stimulated B cells. Vasoactive intestinal peptide (VIP) appears to regulate both IgA , and IgAz production from cultured human B lymphocytes stimulated with anti-CD-40 monoclonal antibodies (Kimata and Fujimoto, 1995). IJsing T-independent modes of bovine B cell activation (SAC and PWM) and a variety of concentrations of recombinant porcine TGF-P. we determined that IgA production was not solely inducible by this factor. Importantly. recombinant porcine TGF-/3 inhibited IgM production from sIgM+ bovine B cells costimulated with PWM and IL-2 without impacting on culture viability. This activity is conserved in LPS-driven murine systems (Coffman et al., 1989; Sonoda et al.. 1989). As TGF-/3 production is not restricted solely to mucosal tissues in species where expression has been examined. it seems unlikely that TGF-/3 alone would explain regional IgA expression. Moreover, TGF-P is largely constitutively expressed by murine B cells irrespective of their state of activation or differentiation (D. Lafrenz, personal communication. 1996). In the mouse. high rate synthesis of IgA is dependent on IL-4 and IL-5 in addition to TGF-/3 (McIntyre et al., 1995). These results are in line with studies in which a preferential T,2-type response is linked to T cells predominantly in the gut (GALT) or mucosa associated lymphoid tissues (MALT) (Fujihashi et al.. 1993). In the regulation of bovine IgM and IgG expression, IL-2 appears to consistently have additive effects if added at the initiation of the culture period and this effect appears to be conserved for IgA production in our studies and those of Collins and Oldham. 1995. In human systems using anti-CD40 monoclonal antibodies, IL-10 also appears to regulate IgA production (Defiance et al.. 1992; Briere et al.. 19941. We therefore tested IL-2 alone and in combination with recombinant human IL- 10 (rhuIL-101 to stimulate IgA production from bovine B cells. RhuIL-10 has been shown in previous studies to be cross-reactive in the bovine (Brown et al., 1994). Although IL-2 and IL-10 alone did not affect IgA production in TGF-P-stimulated cultures with either SAC or PWM using sIgM+ bovine B cells. both cytokines in combination consistently initiated IgA production from in vivo-derived CDS’ B cells but less consistently from CDY bovine B cells.
Acknowledgements Portions 1995.
of this article were reprinted
with permission
from Estes et al.. 1994 and
D.M. Esrrs / Vrterimx~
Immunol~~g?, end Immunoppothol,gy
54 (1996) 61-67
67
References Banchereau, S.. Bazan, F.. Blanchard, D., Briere, F.. Galizzi, S.. van Kooten. C., Liu. Y.. Rousset, F. and Saeland, S., 1994. Ann. Dev. Immunol., 12: 881. Briere, F.. Bridon. J.M., Chevet, D., Sovillet, G., Bienvenu, F., Guret, C., Martinez-Valdez. H. and Banchereau, J., 1994. J. Clin. Invest., 94: 97. Brown. W.C., Woods, V.M.. Chitko-McKown, C., Hash, S. and Rice-Ficht, A., 1994. Infect. Immun., 62: 4697. Butler, J.E.. 1983. Vet. Immunol. Immunopathol., 4: 43. Coffman, R.F., Lebman, D.A. and Shrader, B., 1989. J. Exp. Med., 170: 1039. Collins, R.A. and Oldham, G., 1995. Vet. Immunol. Immunopathol., 44: 141. Defiance. T., Vanbervliet. B.. Briere, F., Durand. I., Rousset, F. and Banchereau, J.. 1992. J. Exp. Med., 175: 682. Estes. D.M.. Glosser, N.M. and Allen, G.K., 1994. Cell Immunol., 154: 287. Estes, D.M.. Glosser. N.M. and Katti, P., 1996. (submitted). Estes, D.M., Hirano, A.. Heussler. V.T.. Dobbelaere, D.A.E. and Brown, W.C., 1995. Cell. Immunol., 16.1: 268. Fujihashi. K.. Yamamoto, M., McGhee, J.R. and Kiyono, H.. 1993. J. Immunol., 151: 6681. Kimata. H. and Fujimoto, M.. 1995. Blood, 85: 2098. Kuhn, R., Rajewsky, K. and Muller, W., 1991. Science, 254: 707. McIntyre, T.M., Kehry, M.R. and Snapper, C.M., 1995. J. Immunol., 154: 3156. Mosmann, T.R. and Coffman, R.L., 1989. Ann. Rev. lmmunol., 7: 145. Noelle, R.J. and Snow, E.C., 1990. Immunol. Today. I I: 361. Purkerson, J.M. and Isakson. P.C., 1992. J. Exp. Med., 175: 973. Snapper, C.M. and Mond, J.J., 1993. Immunol. Today. 14: 15. Snapper, C.M. and Paul, W.E., 1987. J. Immunol., 139: IO. Sonoda, E., Matsumoto, R., Hitoshi, Y., Ishii, T., Sugimoto, M.. Araki, S.. Tominaga, A.. Yamaguchi, N. and Tatatso, K., 1989. J. Exp. Med., 170: 1415. van Vlasselaer, P., Punnonen. J. and de Vries. J.E., 1992. J. Immunol., 148: 2062. Vercelli, D. and Geha, R.S.. 1993. Springer Sem. Immunopathol., 15: 5. Vercelli. D., Jabara, H.H. Arai, K. and Geha, R.S., 1989. J. Exp. Med., 169: 1295.
Veterinary
ELSEVIER
Immunology and Immunopathology 54(1996) 61-67
immunology and immunopathology
Differentiation of B cells in the bovine. Role of cytokines in immunoglobulin isotype expression D. Mark Estes University oj’Missouri,
College
of Veterinwy
Medicine, Depurtment
ofVeterinary
Pathohiology.
Columhiu,
MO 65211. USA
Abstract
Cytokines such as IFN-y, IL-4 and TGF-/3, are known to regulate Ig class switching by positively or negatively regulating germline C, transcription in the mouse and human. Here we show at the protein level that similar regulatory mechanisms are in place in the bovine using various modes of B cell activation and cytokine combinations. Using antigen-receptor cross-linkage or mitogen (pokeweed mitogen) costimulation, we demonstrate that recombinant bovine IL-4 upregulates the expression of IgM, IgG, and IgE in vitro. Upregulation of IgG, and IgE production by rboIL-4 is inhibited in a dose-dependent manner by the addition or rboIFN-y to the cultures. Using similar methodologies, we have also investigated the effects of TGB-P on IgA production in the presence or absence of various cofactors including IL- 10 and IL-2. We find that TGF-/3 will not solely upregulate IgA production from noncommitted precursors (sIgM+) in coculture with mitogen or with forrnalin fixed Staphylococcus cowan Strain I (SAC), but does have positive effects on IgA production in the presence of both IL-2 and IL-IO. These observations suggest that different modes of T-independent costimulation using classical B cell activators does not result in differential IgG subisotype or IgE production by bovine B cells in the presence of cytokines. Together these results suggest that individual cytokines regulate isotype expression in cattle. Keywords:
B cells; Bovine; Cytokines;
Immunoglobin;
Isotope
1. Introduction The bovine immune system differs from other mammalian species with regard to a variety of immune parameters including development of the B cell lineage and the humoral response. For example, cattle appear to lack sIgD and have a unique distribution of immunoglobulin (Ig) isotypes in secretions such as colostrum (Butler, 1983). Due 0165-2427/96/$15.00 PII
Copyright
SO1 65-2427(96)05684-X
0 1996 Elsevier Science B.V. All rights reserved
to fundamental differences in mlmune physiology. cytokine control of antibody production in cattle may differ from the regulatory pathways which have been described in the mouse and human. Recent developments in cell culture systems and the availability of recombinant cytokines have allowed the initial investigations into T cell-B cell interactions. modes of B cell activation/differentiation, and the cytokine regulatory pathways involved in Ig class switching in cattle to begin. Immunoglobulin isotype or class switchin g is the process by which a B lymphocyte can alter expression in Ig heavy chain isotype and express the same VDJ region. altering the effector function of the antibody to a given antigen and thereby expanding the capabilities of the humoral immune response. Class switching in the mammalian species examined to date has been shown to be largely regulated by T cell-derived cytokines and by direct and indirect cognate T cell-B cell interactions (Noelle and Snow. 1990). Studies of T cell-B cell interactions and costimulation have largely focused on the role of the CD40 molecule present on B cells and its counter-receptor on T lymphocytes, the CD40 ligand. These studies have demonstrated that CD40 cross-linking represents the first step in the interaction between T and B lymphocytes and this interaction has profound effects on B cell activation and isotype production (Banchereau et al.. 1994). 7 cell-independent systems of Ig isotype production are suitable for the initial characterization of cytokine regulatory pathways. Cytokines alone are insufficient to effect class switching bv resting B cells and must act in ;I c.ostimulatory or additive fashion with polyclonal B cell activators. For induction of non-IgM isotypes (IgG,, IgG,. IgA. and IgE) in vitro in the bovine. pokeweed mitogen (PWMI with IL-2. SAC, and sheep anti-bovine IgM have been used to support Ig production from resting sIgM’ B cells (Estes et al., 1994; Estes et al.. 1995: Estes et al.. 1996).
2. IFN-7
stimulates
IgG, production
in vitro
Various T-independent costimulation systems have been developed for cytokine costimulation of Ig class switchin g in murine systems in vitro including bacterial lipopolysaccharide (LPSI. polyclonal anti-mouse IgM coupled to agarose beads and anti-IgD dextran (Purkerson and Isakson. 1992; Snapper and Mond. 1993). In the bovine. polyclonal sheep anti-bovine IgM coupled to beads through an avidin-biotin linkage has proven effective in stimulating IgM and non-IgM antibody production on in vitro coculture with crude cytokine containin g culture supernatants or recombinant cytokines (Estes et al.. 1994). Similar results have been reported for PWM with a requirement for IL-2 identified for IgM production in the absence of T cells (Estes et al., 1994; Collins and Oldham. 1995). Resting B cells activated by PWM or sheep anti-bovine IgM coated agarose beads proliferate and upon addition of IFN-y containing supernatants or recombinant bovine IFN-y. secrete mostly IgM and IgG, with no increase in IgG, or IgA levels (Estes et al., 1994). Coculture with recombinant bovine IL-2 potentiated the response by increasing quantitative amounts of Ig produced without qualitatively altering the isotype distribution. Therefore, a predominant T helper cell Type 1 (T,, 1) response in vivo would be implicated in upregulation of IgG, production. In the murine system. T,, I cells have been shown to provide T cell help for IgG?,
D.M. Estes/
Veterinary Immunol~~gy and Immunopathology
54 (1996) 61-67
63
l-
10%
Fig. I. In situ hybridization analysis of anti-p medium alone or 10% T cell culture supematants representative of the percentage of Ig C, region obtained from triplicate cultures for each variable
TCS
stimulated B cells after 48 h reculture in the presence of containing IFN-y in the absence of IL-4 (TCS). Results are mRNA+ cells per 100 total cells as indicated. Cells were and pooled for analysis.
production in vitro (Mosmann and Coffman, 1989). At the transcriptional level, in situ hybridization analysis allows the frequencies of cells producing Ig heavy chain gene segment mRNA for IgG,, IgG, and IgM to be determined on a per cell basis. Analysis of sheep anti-bovine IgM stimulated bovine B lymphocytes at 48 h post-stimulation with IFN-y-containing culture supematants indicated increased frequencies of IgG, mRNA+ cells and reduced frequencies of IgM and IgG, mRNA+ cells relative to control supematants lacking IFN-7 (Fig. 1). These results suggest that IFN-7 controls IgG, production at the transcriptional level.
3. Role of IL-4 in IgG, and IgE production
in cattle
IL-4 and IFN-y in murine systems reciprocally regulate isotype production directly via their actions on B cells and indirectly through T helper cell differentiation pathways leading to T, 1 or T, 2-type responses. In both the mouse and human, IL-4 is integrally involved in the regulation of IgE synthesis and this process can be directed through intermediate switching to IgG, and IgG,, respectively (Vercelli and Geha, 1993). In particular, IgE production was absent in mice rendered deficient for IL-4 production by gene targeting via homologous recombination (Kuhn et al., 1991). In vitro systems which utilize T cells or T cell membranes are complex, therefore we opted to utilize the classical B cell activators (anti-IgM antibodies, SAC, and PWM) to analyze the effects of recombinant bovine IL-4 on Ig isotype production. In these cultures, both PWM and SAC in the presence of rboIL-4 stimulated IgE production in a concentration-dependent manner in vitro from sIgM+ B cells (Fig. 2). These results differ from those obtained with human B cells in which classical activators do not act in concert with IL-4 to stimulate IgE production (Vercelli et al., 1989), again reinforcing the notion that species specificity is an important issue in cytokine regulatory pathways. Consistent with observations in the mouse and human, IL-4 addition to stimulated bovine B cells also resulted in enhanced IgG subclass (IgG,) production (Fig. 2). IgG, production was
64 500
400 T & r300.I e i: 200t I c” TOO.-
N
0
I
%IL~
supernatant added (v/v)
5
v
10
’
20
500
(v/v)
-^ E & 20 c in ‘a 15 r” e 610 w 9
,E
Lm 400 .f E ci
added
25
=‘
2
%IL4 supernatant
300
200
6 c” 100
5
0 %IL~
0 5
10
20
supernatant added (v/v)
Fioc. 2. Fractionated IL--l containing COS cell wpemalanrb uprrgulate cxprcssion of IgM. IpG,. not IgG,
or IgA. (A)
Anti-IgM: SAC + IL2 ml
’ ). IgA
IgM levels (ng ml
’)
in 6 day culture supernalants
( I p*gml ’ ). A, SAC (O.OOS% final concentration); 0. ( I15 II ml ’ ); n (b) IgG, levels (ng ml-- ’ ): CC’) IgG, levels
0,
PWM
and IgE but
as determined by ELISA; PWM + IL2 (ng ml-
’ );
(I 25 U (D)
ml
0,
’ ): A,
IpE levels (ng
levels were equal to medium only cultures under all parameters tested (data not shown). Results
are presented as the mean of triplicate cultures and are representative of three separate experiments from the same donor animal. Standard error values did not exceed 10% of the mean.
induced at relatively lower concentrations than those required for IgE as has been observed in the mouse (Snapper and Mond, 1903). Addition of recombinant bovine IL-2 as a cofactor enhanced synthesis of IgG, and IgE relative to cultures receiving IL-4 only at the same concentrations (Fig. 2). In contrast to LPS-induced murine B lymphocyte cultures receiving IL-4 (Snapper and Paul, 1987). secretory IgM synthesis was also enhanced in a concentration-dependent manner. IgG, and IgE production induced by rboIL-4 were inhibited in a close-dependent manner by IFN-y (Table 11. To further investigate the in vitro activities of IL-4 in the bovine B lineage. the expression of markers known to be affected by IL-4 were also studied in the bovine at 24-48 h following costimulation with classical activators and IL-4 or with IL-4 alone. These markers included IL-2 receptor a-chain (IL-2R). the low affinity IgE receptor CD23, and Class II MHC expression. Flow cytometric analysis of cells cultured for 24 h in the presence of COS-cell derived rboIL-4 alone indicated enhanced expression of all three markers relative to cells cultured in the presence of mock transfected COS cell
D.M. Es!es/
Table 1 Inhibitory
SAC
Immunology
and Immunopathology
effects of IFNy on IgM, IgG,, and IgE secretion
Costimulator Anti-IgM
Veterinary
b
by anti-IgM
Cytokine added
IgM (ng ml- ‘I
0% (v/v) IL-4 20% (v/v) IL-4 20% IL-4+IFNy (100 ng ml-‘) 20%IL-4+IFNy(10ngmll’) 0% (v/v) IL-4 20% (v/v) IL-4 20% IL-4+IFNy (1OOng ml-‘) 20% IL-4+IFNy (lOngml_‘)
llOk9 < 25 43 f 4 < 25 113k12 58+7 82+4
< 25
54 (1996) 61-67
or SAC-stimulated
65
sIgM+
B cells a
IgG, (ng ml- ‘1
IgE (ELISA units) c
18Ok 17 362 f 47 123f8 98k7 125+24 256k51 106+ 19 119f8
< 25 438t4 < 25 < 25 < 25 248 + 33 < 25 < 25
a sIgM+ B cells were obtained from peripheral blood and cultured at 1 X lo5 cells for 6 days and supematants harvested for determination of secreted immunoglobulin levels by ELISA as described in Materials and methods.b biotinylated sheep anti-bovine IgM was added at 1 pg 10e6 cells with avidin coated beads at a 3: 1 bead per cell ratio; SAC was added at 0.005% (v/v) final concentration.c ELISA units ate determined by linear regression relative to a bovine serum standard; data are presented as the mean of triplicate cultures + SEM.
MHC class II
lL2R
CD23
20%114
IL
Frequency
l-
1O%lL4
5%IL4
L
Fluorescence Intensity
c
Fig. 3. Expression of surface IgM, MHC Class II, and IL2R on bovine B cells after 24 h culture with size-fractionated (30 kD > MW > 10 kD) bovine IL-4 from COS cell supematants. Lower panels from bottom to top indicate results obtained by increasing concentrations of IL-4 (O-20%, v/v) in cultures without additional costimulators. Cells were stained with primary antibodies as described in Materials and Methods with mouse anti-bovine MHC Class II (114-B). mouse anti-bovine IL2 receptor (108-A. IL2R). mouse anti-human CD23 (IOB8), murine isotype controls, or biotinylated sheep anti-bovine IgM (The Binding Site) at 1 pg 1O-6 cells. Mouse monoclonal antibodies were detected using biotinylated sheep anti-mouse Ig which had been previously absorbed with a bovine serum agarose column and streptavidin-FITC. Biotinylated sheep primary antibodies were detected using streptavidin-FITC only.
supernatants (Fig. 3). Together these data suggest a broad conservation of the varied activities of IL-4 described for B cells of both mouse and human origin with those in cattle.
4. Regulation
of IgA expression
in the bovine
Transforming growth factor beta (TGF-/?I ) has been established as a switch factor for IgA due to its ability to initiate germline transcripts for the Ig-cu C,, region in the mouse (Coffman et al.. 1989: Sonoda et al.. 19891. TGF-/3 also appears to regulate IgA production in humans (van Vlasselaer et al.. 1992). LPS and PWM were utilized as costimulators for mouse and human B cells in the initial studies of the effects of TGF-,!3, respectively. More recent studies with human B lymphocytes indicate that alternative factors also appear to regulate IgA production utilizing anti-CD40 stimulated B cells. Vasoactive intestinal peptide (VIP) appears to regulate both IgA , and IgAz production from cultured human B lymphocytes stimulated with anti-CD-40 monoclonal antibodies (Kimata and Fujimoto, 1995). IJsing T-independent modes of bovine B cell activation (SAC and PWM) and a variety of concentrations of recombinant porcine TGF-P. we determined that IgA production was not solely inducible by this factor. Importantly. recombinant porcine TGF-/3 inhibited IgM production from sIgM+ bovine B cells costimulated with PWM and IL-2 without impacting on culture viability. This activity is conserved in LPS-driven murine systems (Coffman et al., 1989; Sonoda et al.. 1989). As TGF-/3 production is not restricted solely to mucosal tissues in species where expression has been examined. it seems unlikely that TGF-/3 alone would explain regional IgA expression. Moreover, TGF-P is largely constitutively expressed by murine B cells irrespective of their state of activation or differentiation (D. Lafrenz, personal communication. 1996). In the mouse. high rate synthesis of IgA is dependent on IL-4 and IL-5 in addition to TGF-/3 (McIntyre et al., 1995). These results are in line with studies in which a preferential T,2-type response is linked to T cells predominantly in the gut (GALT) or mucosa associated lymphoid tissues (MALT) (Fujihashi et al.. 1993). In the regulation of bovine IgM and IgG expression, IL-2 appears to consistently have additive effects if added at the initiation of the culture period and this effect appears to be conserved for IgA production in our studies and those of Collins and Oldham. 1995. In human systems using anti-CD40 monoclonal antibodies, IL-10 also appears to regulate IgA production (Defiance et al.. 1992; Briere et al.. 19941. We therefore tested IL-2 alone and in combination with recombinant human IL- 10 (rhuIL-101 to stimulate IgA production from bovine B cells. RhuIL-10 has been shown in previous studies to be cross-reactive in the bovine (Brown et al., 1994). Although IL-2 and IL-10 alone did not affect IgA production in TGF-P-stimulated cultures with either SAC or PWM using sIgM+ bovine B cells. both cytokines in combination consistently initiated IgA production from in vivo-derived CDS’ B cells but less consistently from CDY bovine B cells.
Acknowledgements Portions 1995.
of this article were reprinted
with permission
from Estes et al.. 1994 and
D.M. Esrrs / Vrterimx~
Immunol~~g?, end Immunoppothol,gy
54 (1996) 61-67
67
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