International Immunology, Vol. 4, No. 9, pp. 975 - 983
Transforming growth factor-p1 enhances IgG and IgA sheep red blood cell responses Lenore Zettel1, David A. Clark2, and Peter B. Ernst1 Molecular Virology and Immunology Program and intestinal Disease Research Unit, Departments of Pathology and 2Medicine, McMaster University, 1200 Main Street West, Hamilton, Ontario, Canada L8N 3Z5 Key words: IgA, IgG, sheep red blood cells, transforming growth factor-/31
Antibody responses to ingested antigens can be inhibited by a mechanism known as oral tolerance which acts to prevent excessive stimulation from luminal contents. Local IgA responses can be induced in this non-responsive environment and during intestinal inflammation, mucosal IgG responses can also be increased. The purpose of this study was to compare a panel of cytokines to factors from macrophage - T cell co-culture supernatants for their ability to enhance isotype and sheep red blood cell (SRBC)-specific plaque-forming cell responses in an in vitro model of oral tolerance. IL-2, IL-4, IL-5, and IL-6, which have been implicated in IgA regulation of lipopolysaccharide-stimulated B cells, were not capable of enhancing responses in tolerized cultures; however, transforming growth factor (TGF)-/31 had a dose-dependent ability to enhance responses to the T cell-dependent antigen SRBCs in this system. The enhancement was only seen when antigen was present and was neutralized by specific rabbit antiserum but not normal rabbit IgG. Similar treatment of soluble factors from the macrophage-T cell co-cultures did not inhibit their ability to enhance responses suggesting at least two distinct molecular mechanisms could augment responses in tolerized cultures. This was substantiated further by showing that TGF-/31 was not isotype-specif ic. In contrast, adsorption of the macrophage - T cell co-culture supernatants against monoclonal IgA or IgG removed isotype-specific binding factors which were necessary for the enhancement of IgA and IgG respectively. The cellular target of TGF-/3 in our cultures was the CD8 + suppressor T cells and examination of the kinetics showed TGF-/31 took 2 - 3 days to manifest its activity but was rapidly reversible. Together, these data suggest that TGF-/31 can enhance antigen-driven antibody responses and therefore may contribute to altered responses in inflammation. Introduction In the normal murine intestine, IgA responses predominate which may be partially explained by local helper T cell (TtJ-derived IgA binding factors (1), IL-4 (2), IL-5 (3,4), and IL-6 (5,6). Together, these factors have been implicated in either isotype switch from IgM to IgA (7) or enhancement of antibody production by surface lgA + cells (4,5). Active inhibition of antibody responses (IgM, IgG, IgA, and IgE) is generated by antigen feeding (8-11) but IgA responses at the mucosal surfaces are spared (12,13). The latter may be due to local contrasuppressive mechanism (14) which selectively block IgA-specific suppressor T cells (Tg). In fact, the Vicia w7/osa-adherent contrasuppressor T cells isolated from Peyer's patches have been shown to selectively enhance IgA in orally tolerant mice (10) or in an in vitro model of oral tolerance (15).
We have shown that CD4 + Th produce isotype-specific binding factors that are necessary for the activation of the Vicia villosa adherent contrasuppressor inducer cell which can enhance isotype-specific responses in an in vitro assay of oral tolerance (16). Thus, through the selective effects of isotypespecific binding factors, intestinal IgA responses may become free to react to other cytokines. This model or antibody regulation would allow Th-derived cytokines to focus their bioactivity on IgA enhancement while their pleiotropic effects are prevented. A general property of inflammation in mucosal tissues is the increase in IgG relative to IgA suggesting that local inflammatory cytokines alter antibody isotype regulation. It has been suggested that intestinal inflammation reflects a breakdown in the mechanisms of local tolerance. The objectives of this study were
Correspondence to: P. B. Ernst Transmitting editor: J. W. Schrader
Received 14 October 1991, accepted 27 May 1992
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Abstract
976
TGF-/8 enhances IgA and IgG responses
to screen several different cytokines for their ability to alter antibody isotype in an in vitro model of oral tolerance. In this report we demonstrate that transforming growth factor (TGF)-j31 has the ability to increase both IgG and IgA sheep red blood cell (SRBC)-specific plaque-forming cell (PFC) responses in a system inhibited by Ts, and is distinct from IgA- and IgG-specific factors we have previously described. Thus, the change in the tissue concentration of active TGF-/31 during inflammation may alter the normal pattern of isotype regulation.
Methods Mice
Generation of contrasuppressive factors (CSFs) We and others have previously shown that antigen-pulsed antigen presenting cells co-cultured with CD4 + T cells yield a supernatant that, when combined with a source of Ig (serum or affinity purified Ig) from naive mice, enhances responses in an isotype-specific manner in suppressed cultures (16,17). Briefly, 8 x 105 macrophage-enriched preparations were stimulated by 0.15% SRBCs (National Biological Laboratories, Winnipeg, Canada) in RPMI. Excess antigen was rinsed from the adherent cells and 20 x 107 purified splenic T cells (15,17) were added to the cultures and incubated overnight. These T cell preparations work as well as the CD4 enriched (16). Then, 400 ^ of cell free culture supernatant was mixed with 80 ii\ of normal mouse serum (as a source of Ig) to generate the CSFs (16). Evaluation of antibody responses Single cell suspensions of 20 x 107 spleen cells were stimulated by 0.15% SRBCs in 1 ml minimal essential medium in Marbrook- Diener chambers as previously described (17). The bottom of the 1 ml cell suspension chambers was a semipermeable membrane. Each chamber was suspended in an Erlenmeyer flask containing 100 ml of growth medium to ensure the supply of nutrients and removal of toxins from the growing cells. To suppress these control responses, activated CD8 + Ts were enhanced from tolerant mice and added to the culture (18). In other experiments IgA-specific Ts cells were prepared according to Hoover et al. (19) using the MOPC 315 plasmacytoma which produces IgA (kindly provided by Dr R. Lynch). Briefly, 5 x 106 MOPC 315 tumor cells were put into the peritoneal cavity of syngeneic mice and, following ascites development, CD8 enriched spleen cells were prepared as described elsewhere (16) and 5 x 106 cells were added to the culture. To test for contrasuppressive activity, 60 pi of the CSF from the macrophage - T cell co-culture supernatants combined with Ig as described above was added to the suppressed cultures. In other experiments, we compared regulation by CSFs to other cytokines from Th, including 0.1 - 7 . 0 ng (~1 -100 U/ml)of IL-2
Mechanism of enhancement by TGF-/3 To investigate the mechanism whereby TGF-/31 could increase the B cell responses, TGF-/31 was compared with the CSFs in the macrophage - T cell co-culture supernatants. Rabbit IgG antiTGF-|8 (10 fig; R&D Systems Inc., Minneapolis, MN) or normal rabbit IgG (10 ^g; Sigma, St Louis, MO) were mixed with either factor prior to being added to the assay. To determine if TGF-/31 has Fc binding activity like the CSFs, both TGF-/31 and the co-culture supernatants were passed through a column with monoclonal lgA(TEPC 15), or a mixture containing lgG1 (HDP1), lgG2 (LPL1), lgG2b (MOPC 195), and lgG3 (GAC 8) conjugated to Sepharose. The adherent and non-adherent fractions were collected, concentrated, and tested for functional activity using techniques described elsewhere (16). As an alternate approach, we attempted to determine if TGF-/3 could enhance responses through the release of soluble IgG binding factors. 2.4G2 is a MAb recognizing FC7R (21) and can neutralize IgG responses in our readout system (unpublished observations). Between 0.5 and 5.0 ^g of 2.5G2 was added to cultures treated with TGF-/3. In other experiments, we examined if TGF-/31 acted by activating the splenic responder cell population (RES) or by impairing the function of the Ts. Briefly, 20 x 107 spleen cells were prepared and incubated at 37°C for 1 and 5 h with TGF/31, washed, and used as the RES in an antigen-stimulated suppressed culture. To study the effects of TGF-01 on Ts, cells were enriched from the spleens of mice as described above, then pulsed at 37°C for 1, 3, and 5 min with TGF-/31, washed to remove excess TGF-/3, and added to cultures containing normal, antigen-stimulated RES. TGF-/31 kinetics
To investigate the kinetics, TGF-/31 was added to suppressed cultures on days 0, 1, 2, 3, and 4 using the concentration described above. To study the duration of the effect of TGF-/31, suppressed cultures treated with TGF-/31 that now had enhanced responses were then neutralized with rabbit IgG anti-TGF-/3
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BALB/c (H-2d) mice were obtained from Charles River Laboratories (Wilmington, MA) and CBA/J (H-2k) mice were purchased from Jackson Laboratories (Bar Harbor, ME). All mice were maintained under conventional conditions in our animal facilities at McMaster University and used in these studies at 5 - 1 0 weeks of age.
(Boehringer-Mannheim, Mannheim, Germany), rlL-4 and IL-5 (kindly provided by Dr R. Coffman, DNAX Inc., CA), and IL-6 (kindly provided by Dr J. Gauldie, McMaster University, Canada), as well as 100 pi containing 2.0 ng of purified TGF-/31 derived from porcine platelets (R&D Systems Inc., Minneapolis, MN). Each were individually added to a culture and 5 days later the cells were harvested, and SRBC-specific IgM, IgG, and IgA PFC responses were determined on control, suppressed, and suppressed plus cytokine treated cultures using the Cunningham PFC assay (16). The IgM responses were evaluated as direct plaques, and the IgG and IgA responses were determined indirectly using monospecific rabbit anti-IgG and anti-lgA that were previously tested for specificity by radioimmunoassay (15). IgM responses were subtracted from the indirect PFC values to yield an estimate of IgA and IgG PFC responses. By reading coded slides, it is possible to objectively quantify antigen-specific responses which mimic the magnitude, isotype, and mechanism of regulation of those seen when assaying the spleen of a control or tolerized mouse that has been given an intraperitoneal immunization (10,15,16,20). Values for the PFC were expressed as the number of PFC ± SEM/culture and compared by Student's f-test.
TGF-0 enhances IgA and IgG responses 977 following the same time course. Similar experiments investigating the kinetics of neutralization of the TGF-/?-pulsed Ts were also performed. Results TGF-/31 enhances antibody production in suppressed cultures
The enhancement by CSFs in the co-culture supernatants is unique in that it requires a source of soluble Ig (16). Since a source of exogenous Ig was not required for the TGF-/3, it appeared to be acting by a different mechanism. However, it was possible that CSFs were acting by triggering TGF-/3 release. To test this idea, TGF-/31 or the macrophage - T cell co-culture supernatants plus a source of Ig were added to suppressed cultures with or without neutralizing antiserum against TGF-/3. As shown in Fig. 2, the neutralizing antiserum abrogated the enhancing effect of TGF-/31 but had no statistically significant effect on the activity of control cultures (data not shown) or the CSFs derived from the macrophage-T cell co-culture supernatant. Normal rabbit IgG had no effect on either contrasuppressive activity. In other experiments, synergism between TGF-/31 and the co-culture supernatants was tested by adding both factors to suppressed cultures. The response was not significantly different from suppressed cultures treated with TGF-/31 or co-culture supernatants alone (data not shown). To further compare these two factors, both the macrophage - T cell co-culture supernatants and TGF-/31 were adsorbed on monoclonal IgA or IgG attached to a solid-phase. The data in Fig. 3 show that such an adsorption removed the isotype-specific binding factors from the macrophage-T cell co-cultures and abrogated the enhancement in an isotype-specific manner. However, similar treatment of the TGF-/31 had no effect as all of the TGF-/31 activity remained in the non-adherent fraction. Moreover, attempts to neutralize the IgG binding factor with a mAb (2.4G2) could impair the enhancement by CSFs but had no effect on the TGF-/3 mediated activity (data not shown).
Control
Control + TGF
Ts
Ts + TGF
500
1000
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2000
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3000
3500
SRBC-Specific PFC per Culture
Fig. 1 . Enhancement of IgA and IgG PFC in suppressed cultures by TGF-01. SRBC-specific PFC responses from BALB/c spleen cells stimulated with SRBC in the absence (control) or presence of Ts were compared to TGF-/31 (2.0 ng/ml) treated cultures. PFC responses were tested after 5 days in culture and showed that TGF-/31 could enhance IgG and IgA responses in suppressed systems without having significant effects on control cultures. Results are expressed as the mean of 15 replicates from five separate experiments ± SEM.
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We have shown that supernatants from macrophage-T cell co-cultures containing CSFs enhanced isotype-specific anti-SRBC PFC responses in cultures inhibited by CD8 + Ts (16,17). To compare the function of CSF to other cytokines which function in lipopolysaccharide (LPS)-stimulated B cell assays, we replaced the co-culture supernatants with a panel of purified or recombinant cytokines. The cytokines IL-2, IL-4, IL-5, and IL-6 could not independently mediate any enhancement in suppressed cultures (data not shown); however, as shown in Fig. 1, TGF-/31 mediated a statistically significant enhancement (P < 0.05) of both IgG and IgA responses in suppressed cultures reaching levels comparable to those seen in the control cultures. This effect was dose dependent and the optimal concentrations ranged from 2.0 to 0.4 ng/ml of cultured RES. This effect of a single molecule is in marked contrast to the CSFs in co-culture supernatants which are dependent on separate factors from antigen presenting cells, T cells, and Ig from B cells (16). Similar amounts of TGF-/31 added to control cultures had no affect and, interestingly, did not stimulate IgA production as reported in the LPS-driven system (22,23). TGF-/31 did not enhance the responses unless antigen and Ts were present in the culture, indicating its mechanism of enhancement is dependent on antigen stimulation in a suppressed system.
Mechanism of enhancement by TGF-/S1
978
TGF-fi enhances IgA and IgG responses igM
Control Ts Ts + TGF Ts + TGF + Anti-TGF Ts + Co-culture Sup
500
1000
1500
2OOO
2500
3OOO
3500
SRBC-Specific PFC per Culture Fig. 2. TGF-01 is distinct from isotype-specific CSF. Rabbit anti-TGF-/3 was added to suppressed BALB/c cultures (T^ treated with either TGF-/31 or the CSF in supernatants from macrophage - T cell co-cultures combined with Ig (Co-culture sups). The TGF-/31 activity was specifically neutralized but normal rabbit IgG had no effect. Results are expressed as the mean of six replicates from three separate experiments ± SEM.
Cellular target of TGF-01 To identify the cellular target of TGF-/31 within our culture system, splenic responder cells were cultured with TGF-01 for 60 and 300 min at 37°C before being mixed with Ts and put into culture. Incubation of the RES with TGF-/31 did not enhance responses in a suppressed culture (Fig. 4A). However, when CD8 + Ts were incubated with TGF-/31 (i.e. 150-300 min) there was enhancement of both IgG and IgA responses (Fig. 4B), similar to what is seen when TGF-/31 is added directly to suppressed cultures. Using an isotype-specific suppressor cell which functions by down-regulating Ig heavy chain mRNA (19), we again showed that TGF-/31 inhibited its function (Table 1). Other experiments have shown that incubation of Ts with TGF/3 has no effect on the expression of FcR on the surface of these cells (data not shown). Kinetics of TGF-/S1 and anti-TGF-0 To further explore the mechanism of TGF-/3, the kinetics of TGF-/31 -mediated enhancement of the SRBC responses and the duration of its effects were determined. As shown in the left-hand panel of Fig. 5(A), TGF-/31 acted when added during the first few days of culture. Surprisingly, its effects required the presence of active TGF-/31 throughout the entire time in culture as its neutralization just 24 h before assay totally abrogated any enhancement (right-hand panel, Fig. 5A). This was a specific effect of the anti-TGF-0 as rabbit IgG had no effect on the enhancement by TGF-/31 (data not shown). Similarly, the kinetics of neutralization of the TGF-/j-pulsed Ts followed an identical profile as above (Fig. 5B). The inactivated Ts cells could be reactivated, effectively suppressing both IgG and IgA responses, with the addition of anti-TGF-0 just 24 h prior to determining antibody responses. TGF-/31 functions across genetic restrictions All contrasuppressive function described to date is Igh-restricted (17,24,25). The factors in the macrophage-T cell co-culture
IgA
Control Ts Ts + TGF Ts + CSF + Ig
IgA Adsorbed Ts + TGF AD Ts + TGF NAD Ts + CSF NAD + ig
IgG Adsorbed Ts + TGF AD Ts + TGF NAD Ts + CSF NAD + Ig O
1OOO
2OOO
3OOO
4OOO
SRBC-Specific PFC per Culture Fig. 3. TGF-/31 is not related to isotype-specific binding factors. Further comparison of TGF-/31 and the CSFs in macrophage-T cell co-culture supernatants was done by adsorbing both preparations over IgA or IgG attached to a solid phase. The non-adherent fraction of the TGF-01 had the activity while co-culture supernatants contained isotype-specific binding factors which controlled the isotype of the enhanced response. Results are expressed as the mean of seven replicates from three separate experiments ± SEM.
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+ Anti-TGF
TGF-0 enhances IgA and IgG responses 979
B IgM
IgG
IgA
8000 r
IgG
Itfvl
IgA
60O0
control
Ts 300'
RES 60'
FES 3001
control
Fig. 4. CD8 + Ts is the cellular target of TGF-01. TGF-/31 was incubated for 60 - 3 0 0 min at 37°C with 3 x 106 splenic responder cells (RES) (A) and 2 x 106 CD8 + enriched Ts (B). Following incubations these cells were added to cultures containing SRBCs and untreated Ts (A) or RES (B) and five days later SRBC-specific PFC responses were determined. (A) TGF-01 could not protect the RES from suppression. (B) Prolonged incubation of Ts with TGF-01 (150-300 min), at day 0 of the experiment, inactivated the Ts leading to the enhancement of IgG and IgA responses. Results are expressed as the mean of (A) six replicates from two experiments and (B) 18 replicates from six separate experiments ± SEM.
which initiate contrasuppression have been shown to require an exogenous source of Ig from naive mice that is derived from donors with the same Igh allotype as the T cells used to generate the co-culture supernatant (17). To further compare and contrast TGF-/31 with these factors, experiments were designed to test the effect of TGF-/31 in different strains of mice. The CSFs generated from H-2d cells combined with BALB/c Ig enhanced the response of BALB/c cells but not CBA/J cells (Fig. 6, culture 3). Similarly, supernatant from H-2k cells combined with CBA/J Ig could only enhance the response of the CBA/J cell system (Fig. 6, culture 4). The data in Fig. 6 (cultures 3 - 6 ) show that normal antibody is required but the antibody must be of the appropriate Igh allotype. However, no such antibody was required for enhancement by TGF-01. TGF-/31 amplified IgG and IgA PFC responses in both H-2k and H-2d mice (Fig. 6, culture 7). Therefore, the isotype-specific CSFs in the co-culture supernatants and TGF-/31 appear distinct.
Table 1. TGF-/31 abrogates isotype-specific suppression8 IgM
IgG
IgA
Control
2549 ± 273
1768 ± 463
1102 ± 120
Ts-lgA
4083 ± 427
1221 ± 378
0 ± 0
Ts-lgA + TGF-0
1493 ± 364
594 ± 231
451 ± 29
Ts-lgA + TGF-01 + anti-TGF-/3
1831 ± 406
709 ± 106
0 ± 0
a
SRBC-specific PFC responses from BALB/c spleen cells stimulated with SRBC in the absence (control) or presence of IgA-specific suppressor T cells (Ts-lgA) were compared with TGF-/31 (2.0 ng/ml) treated cultures. PFC responses were tested after 5 days in culture and showed that TGF-01 could enhance IgA responses in an IgA suppressed system. The TGF-01 activity was specifically neutralized by the addition of antiTGF-0 (100 /ig/ml). Results are expressed as the mean of three replicates from a representative experiment ± SEM.
Discussion Recent reports that Peyer's patch derived Vicia villosa adherent cells secrete IL-5 (26) suggest that contrasuppression is in part mediated by the Th2 cells. However, to date, there has not been any evidence that a single molecule can enhance B cell responses in a system where Ts have been activated. The data presented in this paper show that at least two different molecular mechanisms distinct from IL-2, IL-4, IL-5, and IL-6 mediate contrasuppressive-like activity. Only TGF-/31 was sufficient to mediate this effect on its own. TGF-/31 is not responsible for all contrasuppressive activity since it differs from other CSFs. It is neutralized by specific anti-serum but the CSFs in the macrophage - T cell co-cultures are not. In addition, enhancement of IgG by CSFs can be neutralized with a specific mAb to Fc>R (2.4G2) but the effect of TGF-/3 cannot. Previous reports also show that contra-
suppression by other factors is isotype-specific and Igh-restricted (10,16,17,24,25). The source of the Igh restriction appears to be at the level of the Ig needed to interact with the co-culture supernatants since antigen presenting cells and T cells from Igh distinct animals produce functional CSFs as long as the Ig is of the identical Igh allotype as the source of the T cells (17). In contrast to the macrophage - T cell co-culture supernatants, TGF/31 did not have genetic restriction in the experiments reported here. Recent studies show that TGF-/31 can enhance isotype switch in LPS-stimulated B cells to produce IgA which is enhanced even more in the presence of IL-2 and IL-5 (22,23). This may be due to the ability of TGF-/3 to facilitate isotype switch (27,28). These observations suggest TGF-/3 may play a role in the intestine by
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TGF-01 PULSED Ts
PULSED WITH TGF-01
980
TGF-0 enhances IgA and IgG responses TGF-D STIMULATION OF SUPPRESSED CULTURES
ANTI-TGF-0 NEUTRALIZATION OP CONTRASUPPRESSIVE ACTIVITY
Control Ts DAY 0 DAY
1
DAY 2
DAY
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SRBC-Specifc PFC per QJtire
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SFBC-Qpecific PFC per QJtire
IgG
B IgM
6000 Z
5OOO 4OOO 3000 2000
100O O
control
Ts
Ts+TGF01 DAY O
DAY 3
DAY 4
PLUS ANTI-TGF-D Fig. 5. Kinetics of TGF-01 -mediated enhancement. TGF-01 (2 ng/ml) was added to suppressed cultures of BALB/c cells on different days (left-hand panel of A). This molecule only worked when added during the first 3 days of culture, suggesting it acted early or required several days to manifest its activity. When TGF-/J1 was added at the time the cultures were set, either directly to the cultures (right-hand panel of A) or incubated with the Ts (B) and then neutralized on different days (0 - 4) of a 5 day culture, its effect was rapidly neutralized at any time point tested. Results are expressed as the mean of nine replicates from three experiments ± SEM.
facilitating the generation of IgA responses. However, cytokines which work in the LPS-stimulated B cell system to enhance IgA, such as IL-5, do not account for the isotype-specific contrasuppression of IgA since they do not adhere to IgA (2) as seen
with the isotype-specific binding factors (Fig. 3) (16) found in the CSFs. Other cytokines which regulate antibody isotype of LPS-stimulated B cells were tested and did not, on their own, enhance responses in a suppressed system. TGF-/31 was only
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DAY 3
TGF-0 enhances IgA and IgG responses 981 CBA/J Culture
Balb/c Culture
Control
Ts Ts + Balb/c Stp + Balb/c Ig Ts + CBA/J & * + CBA/J Ig Ts + Balb/c Scp + CBA/J Ig Ts + CBA Scp + Balb/c Ig
500
1000
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500
SFBC-Spedfic PFC per Qifcre
IgM
1000
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2500
SflBC-$pedfic PFC per OJftie
IgG
Fig. 6. TGF-/31 is not Igh-restricted. The CSFs in the supernatant from the macrophage-T cell co-cultures (sup) is present only when the cells and source of regulatory Ig are identical at the H2 and Igh locus with the responder cells in the read out system (cultures 3 and 4). Mixing co-culture supernatants made from the cells of one strain with the Ig from the Igh non-identical strain did not generate contrasuppression (cultures 5 and 6). However, TGF-/31 is not H2-restricted and does not require a source of exogenous Ig suggesting it is not Igh-restricted (culture 7). Results are expressed as the mean of three replicates from a representative experiment ± SEM.
active in suppressed cultures in the presence of antigen. These inconsistencies with respect to the effects of TGF-/3 on IgA may be explained by the differences between mitogen- and antigenstimulated systems. In other systems, TGF-/31 has been shown to down-regulate or inactivate many types of immune cells (29-31). Interestingly, TGF-/31 did not inhibit or enhance the T cell-dependent SRBC PFC response in control cultures nor did it alter the responses when responder cells were pulsed before culture suggesting it did not modulate antigen presenting cells, B cells or Th cells. The lack of inhibition may be due to our use of Marbrook - Diener cultures which allows low molecular weight toxins or inhibitory factors to escape into the larger media reservoir. This may be of significance in predicting the effects of TGF-/3 in vivo since blood flow would perfuse tissues and remove inhibitory substances induced by TGF-/3. The Th-derived isotype-specific binding factors have been shown to activate the Vicia w7/osa-adherent contrasuppressor T cells and not the Ts cell (L. Zettel and P. B. Ernst, in preparation). In contrast, incubation of the Ts with TGF-/31 abrogated the suppression while similar treatment of the responder cells had no effect. The type of Ts that was inhibited appears to be related to the isotype-specific suppressors for IgG, IgA, and IgE, whose mechanism of inhibition through FcR has been extensively studied (19,32-35). This suggests that we are studying the regulation of B cells that have already undergone isotype switch. The kinetics of TGF-/31 activity or its neutralization suggested
that the molecule required several days to confer its effect and it must be presented throughout the duration of the culture (Fig. 5A). Moreover, the effect of TGF-/31 on the Ts was rapidly reversible by the addition of specific antiserum after several days in culture. Together these observations suggest that TGF-/3 was carried over on a surface receptor on the Ts cell and that the addition of the antiserum competes for and removes the TGF-/3 which reverses its effect. The fact that most cells express receptors for TGF-/3 but the preincubation of the responders with TGF-/3 was insufficient to mediate the inhibitory effect further supports the hypothesis that the Ts was indeed the specific target. The rapidly reversible effect of anti-TGF-/3 on TGF-/3-treated Ts suggests that TGF-/3 affects the function of Ts and also rules out any cytotoxic effect as the mechanism. TGF-/31 has been shown to inhibit cytotoxic T cell activity mediated by CD8 + cells (36,37). Since TGF-/31 interferes with the suppression mediated by the CD8 + Ts added to our cultures, it is tempting to speculate that TGF-/31 may directly inhibit the suppressive and cytotoxic function of CD8 + cells. Based on reports that enhancement or inhibition of isotype-specific responses can be mediated through soluble binding factors differing only in their degree of glycosylation (38,39), TGF-/3 may modulate the production of these suppressive binding factors, perhaps by increasing their glycosylation. However, we were not able to find any evidence that antibodies to soluble Fc receptors/binding factors could interfere with the enhancement mediated by TGF-/3.
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Ts + TGF
982
TGF-0 enhances IgA and IgG responses
Acknowledgements The authors wish to thank Drs R. Lynch, L. Mayer, and K. Croitoru for their comments and suggestions. This work was supported by grants from the Medical Research Council of Canada. D.A.C. is a recipient of an MRC Scientist award. Abbreviations CSF LPS PFC RES SRBC TGF-/31 Th Ts
contrasuppressive factors lipopolysaccharide plaque-forming cells responder cell population sheep red blood cell transforming growth factor-/31 helper T cells suppressor T cells
References 1 Kiyono, H., Mosteller-Barnum, L. M., Pitts, A. M., Williamson, S. I., Michalek, S. M., and McGhee, J. R. 1985. Isotype-specific immunoregulation. IgA-binding factors produced by Fca receptorpositive T cell hybridomas regulate IgA responses. J. Exp. Med. 161:731. 2 Murray, P. D., McKenzie, D. T., Swain, S. L, and Kagnoff, M. F. 1987. Interleukin 5 and interleukin 4 produced by Peyer's patch T cells selectively enhance immunoglobulin A expression. J. Immunol. 139:2669. 3 Coffman, R. L , Shrader, B., Carty, J., Mosmann, T. R., and Bond, M. W. 1987. A mouse T cell produce that preferentially enhances IgA production I. Biologic characterization. J. Immunol. 139:3685. 4 Beagtey, K. W., Eldridge, J. H., Kiyono, H., Everson, M. P., Koopman, W. J., Honjo, T., and McGhee, J. R. 1988. Recombinant murine IL-5 induces high rate IgA synthesis in cycling IgA-positive Peyer's patch 8 cells. J. Immunol. 141:2035. 5 Beagley, K. W., Eldridge, J. H., Lee, F., Kiyono, H., Everson, M. P., Koopman, W. J., Hirano, T., Kishimoto, T., and McGhee, J. R. 1989. Interieukins and IgA synthesis. Human and murine interleukin 6 induce high rate IgA secretion in IgA-committed B cells. J. Exp. Med. 169:2133. 6 Kunimoto, D. Y., Nordan, R. P., and Strober, W. 1989. II-6 is a potent
cofactor of IL-1 in IgM synthesis and of IL-5 in IgA synthesis. J. Immunol. 143:2230. 7 Kunimoto, D. Y., Harriman, G. R., and Strober, W. 1988. Regulation of IgA differentiation in CH12LX B cells by lymphokines: IL-4 induces membrane IgM-positive CH12LX cells to express membrane IgA and II-5 induces membrane IgA-positive CH12LX cells to secrete IgA. J. Immunol. 141:713. 8 Mattingly, J. A. and Waksman, B. H. 1978. Immunologic suppression after oral administration of antigen. I. Specific suppressor cells formed in rat Peyer's patches after oral administration of sheep erythrocytes and their systemic migration. J. Immunol. 121:1878. 9 Ngan, J. and Kind, L. S. 1978. Suppressor T cells for IgE and IgG in Peyer's patches of mice made tolerant by the oral administration of ovalbumin. J. Immunol. 120:861. 10 Suzuki, I., Kitamura, K., Kiyono, H., Kurita, T., Green, D. R., and McGhee, J. R. 1986. Isotype-specific immunoregulation. Evidence for a distinct subset of T contrasuppressor cells for IgA responses in murine Peyer's patches. J. Exp. Med. 164:501. 11 Kitamura, K., Kiyono, H., Fijihashi, K., Eldridge, J. H., Green, D. R., and McGhee, J. R. 1987. Contrasuppressor cells that break oral tolerance are antigen-specific T cells distinct from T helper (L3T4), T suppressor (Lyt-2+) and B cells. J. Immunol. 139:3251. 12 Challacombe, S. J. and Tomasi, T. B. 1980. Systemic tolerance and secretory immunity after oral immunization. J. Exp. Med. 152:1459. 13 Richman, L. K., Graeff, A. S., Yarchoan, R., and Strober, W. 1981. Simultaneous induction of antigen-specific IgA helper T cells and IgG suppressor T cells in the murine Peyer's patch after protein feeding. J. Immunol. 121:2429. 14 Green, D. R., Gold, J., Martin, S., Gershon, R., and Gershon, R. K. 1982. Microenvironmental immunoregulation: possible role of contrasuppressor cells in maintaining immune responses in gutassociated lymphoid tissue. Proc. Natl Acad. Sci. USA 79:889. 15 Ernst, P. B., Maeba, J., Lee, S.-T., and Paraskevas, F. 1988. A novel mechanism for the selection of isotype-specific antibody responses: the role of intestinal T cells in the regulation of IgA synthesis by the anti-suppressor circuit. Immunology 65:59. 16 Ernst, P. B., Lee, S.-T., Maeba, J., Stanisz, A. M., and Paraskevas, F. 1989. A role for isotype-specific binding factors in the regulation of IgA- and IgG-specific responses by the anti/contrasuppressor T cell circuit. J. Immunol. 143:1426. 17 Maeba, J., Lee, S.-T., and Paraskevas, F. 1988. Macrophage T cell interaction. Induction in vitro of a mediator with potent antisuppressor activity. J. Immunol. Methods 106:7. 18 Perry, L. L. and Greene, M. I. 1982. Conversion of immunity to suppression by in vivo administration of I-A subregion-specific antibodies. J. Exp. Med. 480:491. 19 Hoover, R. G., Dieckgraefe, B. K., and Lynch, R. G. 1981. T cells with Fc Receptors for IgA: induction of Ta cells in vivo and in vitro by purified IgA. J. Immunol. 127:1560. 20 Suzuki, I., Kiyono, H., Kitamura, K., Green, D. R., and McGhee, J. R. 1986. Abrogation of oral tolerance by contrasuppressor T cells suggests the presence of regulatory T-cell networks in the mucosal immune system. Nature 320:451. 21 Unkeless, J. C. 1979. Characterization of a monoclonal antibody directed against mouse macrophage and lymphocyte Fc gamma receptors. J. Exp. Med. 167:580. 22 Sonoda, E., Matsumoto, R., Hitoshi, Y., Ishil, T., Sugimoto, M., Araki, S., Tominaga, A., Yamaguchi, N., and Takatsu, K. 1989. Transforming growth factor B induces IgA production and acts additively with interteukin 5 for IgA production. J. Exp. Med. 170:1415. 23 Coffman, R. L., Lebman, D. A., and Shrader, B. 1989. Transforming growth factor beta specifically enhances IgA production by lipopolysaccharide-stimulated murine B lymphocytes. J. Exp. Med. 170:1039. 24 Braley-Mullen, H. 1986. Characterization and activity of contrasuppressor T cells induced by type III pneumococcal polysaccharide. J. Immunol. 137:2761. 25 Green, D. R., Chue, B., and Flood, P. M. 1988. Induction of contrasuppression is restricted by genes mapping to the Igh locus. Immunol. Res. 7:82. 26 Schoenbeck, S., Hammen, M. J., and Kagnoff, M. F. 1989. Vicia villosa agglutinin separates freshly isolated Peyer's Patch T cells into interleukin 5- or interleukin 2-producing subsets. J. Exp. Med. 169:1491.
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In face of the tremendous antigenic onslaught from the intestinal lumen, the down-regulation of pro-inflammatory antibodies such as the C'-fixing IgM and IgG, and the allergy-associated IgE is desirable. Since IgA does not efficiently fix C by the classical pathway (40), this isotype is desirable in the intestine as it is transported into the mucus where it performs its role of antigen exclusion. In inflammatory bowel disease, the isotype regulation is disrupted with IgG responses being much more prevalent (41). One explanation is that the orchestration of antibody responses by regulatory T cells is perturbed. The data in this paper suggest TGF-/3 may indiscriminantly enhance IgG and IgA as an alternative mechanism to account for the elevated IgG responses in the inflamed intestine. TGF-/3 is produced in many inflammatory situations (42) including the inflamed murine or human intestine (L. Zettel and P. B. Ernst, unpublished observations). It is associated with healing and repair mechanisms (43) and can be produced by T cells during the normal immune response. A role for TGF-/3 in promoting inflammation is supported by the recent observation that TGF-/3 can induce inflammation on its own when injected into joints (44). Future studies should elucidate the role of TGF-/3 in promoting IgG production in the inflamed mucosal environment.
TGF-0 enhances IgA and IgG responses 983 36 Mule, J. J., Schwarz, S. L., Roberts, A. B., Sporn, M. B., and Rosenberg, S. A. 1988. Transforming growth factor-beta inhibits the in vitro generation of lymphokine-activated killer cells and cytotoxic T cells. Cancer Immunol. Immunother. 26:95. 37 Shalaby, M. R. and Ammann, A. J. 1988. Suppression of immune cell function in vitro by recombinant human transforming growth factorB. Cell. Immunol. 112:343. 38 Yodoi, J., Hirashima, M., and Ishizaka, K. 1982. Regulatory role of IgE-binding factors from rat T lymphocytes. V. The carbohydrate moieties in IgE-potentiating factors and IgE-suppressive factors. J. Immunol. 128:289. 39 Ishizaka, K. 1988. IgE-binding factors and regulation of the IgE antibody response. Annu. Rev. Immunol. 6:513. 40 Pfaffenbach, G., Lamm, M. E., and Gigli, I. 1982. Activation of the guinea pig alternative complement pathway by mouse IgA immune complexes. J. Exp. Med. 155:231. 41 MacDermott, R. P., Nash, G. S., Bertovich, M. J., Seiden, M. V., Bragdon, M. J., and Beale, M. G. 1981. Alterations of IgM, IgG, and IgA synthesis and secretion by peripheral blood and intestinal mononuclear cells from patients with ulcerative colitis and Crohn's disease. Gastroenterology 81:844. 42 Wahl, S. M., McCartney-Francis, N., and Mergenhagen, S. E. 1989. Inflammatory and immunomodulatory roles of TGF-j3. Immunol. Today 10:258. 43 Roberts, A. B., Shull, J. H., Smith, J. M., and Ward, J. M. 1983. Polypeptide transforming growth factors isolated from bovine sources and used for wound healing. Science 219:1329. 44 Allen, J. B., Manthey, C. L , Hand, A. R., Ohura, K., Ellingsworth, L , and Wahl, S. M. 1990. Rapid onset synovial inflammation and hyperplasia induced by transforming growth factor beta. J. Exp. Med. 171:231.
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27 Lebman, D. A., Nomura, D. Y., Coffman, R. L, and Lee, F. D. 1990. Molecular characterization of germ-line immunoglobulin A transcripts producing during transforming growth factor type /3-induced isotype switching. Proc. Natl Acad. Sci. USA 87:3962. 28 Kim. P.-H. and Kagnoff, M. F. 1990. Transforming growth factor 01 increases IgA isotype switching at the clonal level. J. Immunol. 145:3773. 29 Kehrl, J. H., Wakefield, L. M., Roberts, A. B., Jakowlew, S., Alvarez-Mon, M., Derynck, R., Sporn, M. B., and Fauci, A. S. 1986. Production of transforming growth factor /3 by human T lymphocytes and its potential role in the regulation of T cell growth. J. Exp. Med. 163:1037. 30 Roberts, A. B., Anzano, M. A., Wakefield, L. M., Roche, N. S., Stern, D. F., and Sporn, M. B. 1985. Type 0 transforming growth factor: a bifunctional regulator of cellular growth. Proc. Natl. Acad. Sci. USA 82:119. 31 Sporn, M. B., Roberts, A. B., Wakefield, L. M., and Assoian, R. K. 1986. Transforming growth factor-0: biological function and chemical structure. Science 233:532. 32 Hoover, R. G. and Lynch, R. G. 1983. Isotype-specific suppression of IgA: suppression of IgA responses in BALB/c mice by T cells. J. Immunol. 130:521. 33 Huff, T. F., Uede, T., Iwata, M., and Ishizaka, K. 1983. Modulation of the biologic activities of IgE-binding factors. III. Switching of a T cell hybrid clone from the formation of IgE-suppressive factor to the formation of IgE-potentiating factor. J. Immunol. 131:1090. 34 Lynch, R. G. 1987. Immunoglobulin-specific suppressor T cells. Adv. Immunol. 40:135. 35 Varin, N., Sautes, C , Even, J., Hogarth, P. M., and Fridman, W. H. 1989. Recombinant soluble receptors of the FCT portion inhibit antibody production in vitro. Eur J. Immunol. 19:2263.
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Transforming growth factor-p1 enhances IgG and IgA sheep red blood cell responses Lenore Zettel1, David A. Clark2, and Peter B. Ernst1 Molecular Virology and Immunology Program and intestinal Disease Research Unit, Departments of Pathology and 2Medicine, McMaster University, 1200 Main Street West, Hamilton, Ontario, Canada L8N 3Z5 Key words: IgA, IgG, sheep red blood cells, transforming growth factor-/31
Antibody responses to ingested antigens can be inhibited by a mechanism known as oral tolerance which acts to prevent excessive stimulation from luminal contents. Local IgA responses can be induced in this non-responsive environment and during intestinal inflammation, mucosal IgG responses can also be increased. The purpose of this study was to compare a panel of cytokines to factors from macrophage - T cell co-culture supernatants for their ability to enhance isotype and sheep red blood cell (SRBC)-specific plaque-forming cell responses in an in vitro model of oral tolerance. IL-2, IL-4, IL-5, and IL-6, which have been implicated in IgA regulation of lipopolysaccharide-stimulated B cells, were not capable of enhancing responses in tolerized cultures; however, transforming growth factor (TGF)-/31 had a dose-dependent ability to enhance responses to the T cell-dependent antigen SRBCs in this system. The enhancement was only seen when antigen was present and was neutralized by specific rabbit antiserum but not normal rabbit IgG. Similar treatment of soluble factors from the macrophage-T cell co-cultures did not inhibit their ability to enhance responses suggesting at least two distinct molecular mechanisms could augment responses in tolerized cultures. This was substantiated further by showing that TGF-/31 was not isotype-specif ic. In contrast, adsorption of the macrophage - T cell co-culture supernatants against monoclonal IgA or IgG removed isotype-specific binding factors which were necessary for the enhancement of IgA and IgG respectively. The cellular target of TGF-/3 in our cultures was the CD8 + suppressor T cells and examination of the kinetics showed TGF-/31 took 2 - 3 days to manifest its activity but was rapidly reversible. Together, these data suggest that TGF-/31 can enhance antigen-driven antibody responses and therefore may contribute to altered responses in inflammation. Introduction In the normal murine intestine, IgA responses predominate which may be partially explained by local helper T cell (TtJ-derived IgA binding factors (1), IL-4 (2), IL-5 (3,4), and IL-6 (5,6). Together, these factors have been implicated in either isotype switch from IgM to IgA (7) or enhancement of antibody production by surface lgA + cells (4,5). Active inhibition of antibody responses (IgM, IgG, IgA, and IgE) is generated by antigen feeding (8-11) but IgA responses at the mucosal surfaces are spared (12,13). The latter may be due to local contrasuppressive mechanism (14) which selectively block IgA-specific suppressor T cells (Tg). In fact, the Vicia w7/osa-adherent contrasuppressor T cells isolated from Peyer's patches have been shown to selectively enhance IgA in orally tolerant mice (10) or in an in vitro model of oral tolerance (15).
We have shown that CD4 + Th produce isotype-specific binding factors that are necessary for the activation of the Vicia villosa adherent contrasuppressor inducer cell which can enhance isotype-specific responses in an in vitro assay of oral tolerance (16). Thus, through the selective effects of isotypespecific binding factors, intestinal IgA responses may become free to react to other cytokines. This model or antibody regulation would allow Th-derived cytokines to focus their bioactivity on IgA enhancement while their pleiotropic effects are prevented. A general property of inflammation in mucosal tissues is the increase in IgG relative to IgA suggesting that local inflammatory cytokines alter antibody isotype regulation. It has been suggested that intestinal inflammation reflects a breakdown in the mechanisms of local tolerance. The objectives of this study were
Correspondence to: P. B. Ernst Transmitting editor: J. W. Schrader
Received 14 October 1991, accepted 27 May 1992
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Abstract
976
TGF-/8 enhances IgA and IgG responses
to screen several different cytokines for their ability to alter antibody isotype in an in vitro model of oral tolerance. In this report we demonstrate that transforming growth factor (TGF)-j31 has the ability to increase both IgG and IgA sheep red blood cell (SRBC)-specific plaque-forming cell (PFC) responses in a system inhibited by Ts, and is distinct from IgA- and IgG-specific factors we have previously described. Thus, the change in the tissue concentration of active TGF-/31 during inflammation may alter the normal pattern of isotype regulation.
Methods Mice
Generation of contrasuppressive factors (CSFs) We and others have previously shown that antigen-pulsed antigen presenting cells co-cultured with CD4 + T cells yield a supernatant that, when combined with a source of Ig (serum or affinity purified Ig) from naive mice, enhances responses in an isotype-specific manner in suppressed cultures (16,17). Briefly, 8 x 105 macrophage-enriched preparations were stimulated by 0.15% SRBCs (National Biological Laboratories, Winnipeg, Canada) in RPMI. Excess antigen was rinsed from the adherent cells and 20 x 107 purified splenic T cells (15,17) were added to the cultures and incubated overnight. These T cell preparations work as well as the CD4 enriched (16). Then, 400 ^ of cell free culture supernatant was mixed with 80 ii\ of normal mouse serum (as a source of Ig) to generate the CSFs (16). Evaluation of antibody responses Single cell suspensions of 20 x 107 spleen cells were stimulated by 0.15% SRBCs in 1 ml minimal essential medium in Marbrook- Diener chambers as previously described (17). The bottom of the 1 ml cell suspension chambers was a semipermeable membrane. Each chamber was suspended in an Erlenmeyer flask containing 100 ml of growth medium to ensure the supply of nutrients and removal of toxins from the growing cells. To suppress these control responses, activated CD8 + Ts were enhanced from tolerant mice and added to the culture (18). In other experiments IgA-specific Ts cells were prepared according to Hoover et al. (19) using the MOPC 315 plasmacytoma which produces IgA (kindly provided by Dr R. Lynch). Briefly, 5 x 106 MOPC 315 tumor cells were put into the peritoneal cavity of syngeneic mice and, following ascites development, CD8 enriched spleen cells were prepared as described elsewhere (16) and 5 x 106 cells were added to the culture. To test for contrasuppressive activity, 60 pi of the CSF from the macrophage - T cell co-culture supernatants combined with Ig as described above was added to the suppressed cultures. In other experiments, we compared regulation by CSFs to other cytokines from Th, including 0.1 - 7 . 0 ng (~1 -100 U/ml)of IL-2
Mechanism of enhancement by TGF-/3 To investigate the mechanism whereby TGF-/31 could increase the B cell responses, TGF-/31 was compared with the CSFs in the macrophage - T cell co-culture supernatants. Rabbit IgG antiTGF-|8 (10 fig; R&D Systems Inc., Minneapolis, MN) or normal rabbit IgG (10 ^g; Sigma, St Louis, MO) were mixed with either factor prior to being added to the assay. To determine if TGF-/31 has Fc binding activity like the CSFs, both TGF-/31 and the co-culture supernatants were passed through a column with monoclonal lgA(TEPC 15), or a mixture containing lgG1 (HDP1), lgG2 (LPL1), lgG2b (MOPC 195), and lgG3 (GAC 8) conjugated to Sepharose. The adherent and non-adherent fractions were collected, concentrated, and tested for functional activity using techniques described elsewhere (16). As an alternate approach, we attempted to determine if TGF-/3 could enhance responses through the release of soluble IgG binding factors. 2.4G2 is a MAb recognizing FC7R (21) and can neutralize IgG responses in our readout system (unpublished observations). Between 0.5 and 5.0 ^g of 2.5G2 was added to cultures treated with TGF-/3. In other experiments, we examined if TGF-/31 acted by activating the splenic responder cell population (RES) or by impairing the function of the Ts. Briefly, 20 x 107 spleen cells were prepared and incubated at 37°C for 1 and 5 h with TGF/31, washed, and used as the RES in an antigen-stimulated suppressed culture. To study the effects of TGF-01 on Ts, cells were enriched from the spleens of mice as described above, then pulsed at 37°C for 1, 3, and 5 min with TGF-/31, washed to remove excess TGF-/3, and added to cultures containing normal, antigen-stimulated RES. TGF-/31 kinetics
To investigate the kinetics, TGF-/31 was added to suppressed cultures on days 0, 1, 2, 3, and 4 using the concentration described above. To study the duration of the effect of TGF-/31, suppressed cultures treated with TGF-/31 that now had enhanced responses were then neutralized with rabbit IgG anti-TGF-/3
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BALB/c (H-2d) mice were obtained from Charles River Laboratories (Wilmington, MA) and CBA/J (H-2k) mice were purchased from Jackson Laboratories (Bar Harbor, ME). All mice were maintained under conventional conditions in our animal facilities at McMaster University and used in these studies at 5 - 1 0 weeks of age.
(Boehringer-Mannheim, Mannheim, Germany), rlL-4 and IL-5 (kindly provided by Dr R. Coffman, DNAX Inc., CA), and IL-6 (kindly provided by Dr J. Gauldie, McMaster University, Canada), as well as 100 pi containing 2.0 ng of purified TGF-/31 derived from porcine platelets (R&D Systems Inc., Minneapolis, MN). Each were individually added to a culture and 5 days later the cells were harvested, and SRBC-specific IgM, IgG, and IgA PFC responses were determined on control, suppressed, and suppressed plus cytokine treated cultures using the Cunningham PFC assay (16). The IgM responses were evaluated as direct plaques, and the IgG and IgA responses were determined indirectly using monospecific rabbit anti-IgG and anti-lgA that were previously tested for specificity by radioimmunoassay (15). IgM responses were subtracted from the indirect PFC values to yield an estimate of IgA and IgG PFC responses. By reading coded slides, it is possible to objectively quantify antigen-specific responses which mimic the magnitude, isotype, and mechanism of regulation of those seen when assaying the spleen of a control or tolerized mouse that has been given an intraperitoneal immunization (10,15,16,20). Values for the PFC were expressed as the number of PFC ± SEM/culture and compared by Student's f-test.
TGF-0 enhances IgA and IgG responses 977 following the same time course. Similar experiments investigating the kinetics of neutralization of the TGF-/?-pulsed Ts were also performed. Results TGF-/31 enhances antibody production in suppressed cultures
The enhancement by CSFs in the co-culture supernatants is unique in that it requires a source of soluble Ig (16). Since a source of exogenous Ig was not required for the TGF-/3, it appeared to be acting by a different mechanism. However, it was possible that CSFs were acting by triggering TGF-/3 release. To test this idea, TGF-/31 or the macrophage - T cell co-culture supernatants plus a source of Ig were added to suppressed cultures with or without neutralizing antiserum against TGF-/3. As shown in Fig. 2, the neutralizing antiserum abrogated the enhancing effect of TGF-/31 but had no statistically significant effect on the activity of control cultures (data not shown) or the CSFs derived from the macrophage-T cell co-culture supernatant. Normal rabbit IgG had no effect on either contrasuppressive activity. In other experiments, synergism between TGF-/31 and the co-culture supernatants was tested by adding both factors to suppressed cultures. The response was not significantly different from suppressed cultures treated with TGF-/31 or co-culture supernatants alone (data not shown). To further compare these two factors, both the macrophage - T cell co-culture supernatants and TGF-/31 were adsorbed on monoclonal IgA or IgG attached to a solid-phase. The data in Fig. 3 show that such an adsorption removed the isotype-specific binding factors from the macrophage-T cell co-cultures and abrogated the enhancement in an isotype-specific manner. However, similar treatment of the TGF-/31 had no effect as all of the TGF-/31 activity remained in the non-adherent fraction. Moreover, attempts to neutralize the IgG binding factor with a mAb (2.4G2) could impair the enhancement by CSFs but had no effect on the TGF-/3 mediated activity (data not shown).
Control
Control + TGF
Ts
Ts + TGF
500
1000
1500
2000
2500
3000
3500
SRBC-Specific PFC per Culture
Fig. 1 . Enhancement of IgA and IgG PFC in suppressed cultures by TGF-01. SRBC-specific PFC responses from BALB/c spleen cells stimulated with SRBC in the absence (control) or presence of Ts were compared to TGF-/31 (2.0 ng/ml) treated cultures. PFC responses were tested after 5 days in culture and showed that TGF-/31 could enhance IgG and IgA responses in suppressed systems without having significant effects on control cultures. Results are expressed as the mean of 15 replicates from five separate experiments ± SEM.
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We have shown that supernatants from macrophage-T cell co-cultures containing CSFs enhanced isotype-specific anti-SRBC PFC responses in cultures inhibited by CD8 + Ts (16,17). To compare the function of CSF to other cytokines which function in lipopolysaccharide (LPS)-stimulated B cell assays, we replaced the co-culture supernatants with a panel of purified or recombinant cytokines. The cytokines IL-2, IL-4, IL-5, and IL-6 could not independently mediate any enhancement in suppressed cultures (data not shown); however, as shown in Fig. 1, TGF-/31 mediated a statistically significant enhancement (P < 0.05) of both IgG and IgA responses in suppressed cultures reaching levels comparable to those seen in the control cultures. This effect was dose dependent and the optimal concentrations ranged from 2.0 to 0.4 ng/ml of cultured RES. This effect of a single molecule is in marked contrast to the CSFs in co-culture supernatants which are dependent on separate factors from antigen presenting cells, T cells, and Ig from B cells (16). Similar amounts of TGF-/31 added to control cultures had no affect and, interestingly, did not stimulate IgA production as reported in the LPS-driven system (22,23). TGF-/31 did not enhance the responses unless antigen and Ts were present in the culture, indicating its mechanism of enhancement is dependent on antigen stimulation in a suppressed system.
Mechanism of enhancement by TGF-/S1
978
TGF-fi enhances IgA and IgG responses igM
Control Ts Ts + TGF Ts + TGF + Anti-TGF Ts + Co-culture Sup
500
1000
1500
2OOO
2500
3OOO
3500
SRBC-Specific PFC per Culture Fig. 2. TGF-01 is distinct from isotype-specific CSF. Rabbit anti-TGF-/3 was added to suppressed BALB/c cultures (T^ treated with either TGF-/31 or the CSF in supernatants from macrophage - T cell co-cultures combined with Ig (Co-culture sups). The TGF-/31 activity was specifically neutralized but normal rabbit IgG had no effect. Results are expressed as the mean of six replicates from three separate experiments ± SEM.
Cellular target of TGF-01 To identify the cellular target of TGF-/31 within our culture system, splenic responder cells were cultured with TGF-01 for 60 and 300 min at 37°C before being mixed with Ts and put into culture. Incubation of the RES with TGF-/31 did not enhance responses in a suppressed culture (Fig. 4A). However, when CD8 + Ts were incubated with TGF-/31 (i.e. 150-300 min) there was enhancement of both IgG and IgA responses (Fig. 4B), similar to what is seen when TGF-/31 is added directly to suppressed cultures. Using an isotype-specific suppressor cell which functions by down-regulating Ig heavy chain mRNA (19), we again showed that TGF-/31 inhibited its function (Table 1). Other experiments have shown that incubation of Ts with TGF/3 has no effect on the expression of FcR on the surface of these cells (data not shown). Kinetics of TGF-/S1 and anti-TGF-0 To further explore the mechanism of TGF-/3, the kinetics of TGF-/31 -mediated enhancement of the SRBC responses and the duration of its effects were determined. As shown in the left-hand panel of Fig. 5(A), TGF-/31 acted when added during the first few days of culture. Surprisingly, its effects required the presence of active TGF-/31 throughout the entire time in culture as its neutralization just 24 h before assay totally abrogated any enhancement (right-hand panel, Fig. 5A). This was a specific effect of the anti-TGF-0 as rabbit IgG had no effect on the enhancement by TGF-/31 (data not shown). Similarly, the kinetics of neutralization of the TGF-/j-pulsed Ts followed an identical profile as above (Fig. 5B). The inactivated Ts cells could be reactivated, effectively suppressing both IgG and IgA responses, with the addition of anti-TGF-0 just 24 h prior to determining antibody responses. TGF-/31 functions across genetic restrictions All contrasuppressive function described to date is Igh-restricted (17,24,25). The factors in the macrophage-T cell co-culture
IgA
Control Ts Ts + TGF Ts + CSF + Ig
IgA Adsorbed Ts + TGF AD Ts + TGF NAD Ts + CSF NAD + ig
IgG Adsorbed Ts + TGF AD Ts + TGF NAD Ts + CSF NAD + Ig O
1OOO
2OOO
3OOO
4OOO
SRBC-Specific PFC per Culture Fig. 3. TGF-/31 is not related to isotype-specific binding factors. Further comparison of TGF-/31 and the CSFs in macrophage-T cell co-culture supernatants was done by adsorbing both preparations over IgA or IgG attached to a solid phase. The non-adherent fraction of the TGF-01 had the activity while co-culture supernatants contained isotype-specific binding factors which controlled the isotype of the enhanced response. Results are expressed as the mean of seven replicates from three separate experiments ± SEM.
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+ Anti-TGF
TGF-0 enhances IgA and IgG responses 979
B IgM
IgG
IgA
8000 r
IgG
Itfvl
IgA
60O0
control
Ts 300'
RES 60'
FES 3001
control
Fig. 4. CD8 + Ts is the cellular target of TGF-01. TGF-/31 was incubated for 60 - 3 0 0 min at 37°C with 3 x 106 splenic responder cells (RES) (A) and 2 x 106 CD8 + enriched Ts (B). Following incubations these cells were added to cultures containing SRBCs and untreated Ts (A) or RES (B) and five days later SRBC-specific PFC responses were determined. (A) TGF-01 could not protect the RES from suppression. (B) Prolonged incubation of Ts with TGF-01 (150-300 min), at day 0 of the experiment, inactivated the Ts leading to the enhancement of IgG and IgA responses. Results are expressed as the mean of (A) six replicates from two experiments and (B) 18 replicates from six separate experiments ± SEM.
which initiate contrasuppression have been shown to require an exogenous source of Ig from naive mice that is derived from donors with the same Igh allotype as the T cells used to generate the co-culture supernatant (17). To further compare and contrast TGF-/31 with these factors, experiments were designed to test the effect of TGF-/31 in different strains of mice. The CSFs generated from H-2d cells combined with BALB/c Ig enhanced the response of BALB/c cells but not CBA/J cells (Fig. 6, culture 3). Similarly, supernatant from H-2k cells combined with CBA/J Ig could only enhance the response of the CBA/J cell system (Fig. 6, culture 4). The data in Fig. 6 (cultures 3 - 6 ) show that normal antibody is required but the antibody must be of the appropriate Igh allotype. However, no such antibody was required for enhancement by TGF-01. TGF-/31 amplified IgG and IgA PFC responses in both H-2k and H-2d mice (Fig. 6, culture 7). Therefore, the isotype-specific CSFs in the co-culture supernatants and TGF-/31 appear distinct.
Table 1. TGF-/31 abrogates isotype-specific suppression8 IgM
IgG
IgA
Control
2549 ± 273
1768 ± 463
1102 ± 120
Ts-lgA
4083 ± 427
1221 ± 378
0 ± 0
Ts-lgA + TGF-0
1493 ± 364
594 ± 231
451 ± 29
Ts-lgA + TGF-01 + anti-TGF-/3
1831 ± 406
709 ± 106
0 ± 0
a
SRBC-specific PFC responses from BALB/c spleen cells stimulated with SRBC in the absence (control) or presence of IgA-specific suppressor T cells (Ts-lgA) were compared with TGF-/31 (2.0 ng/ml) treated cultures. PFC responses were tested after 5 days in culture and showed that TGF-01 could enhance IgA responses in an IgA suppressed system. The TGF-01 activity was specifically neutralized by the addition of antiTGF-0 (100 /ig/ml). Results are expressed as the mean of three replicates from a representative experiment ± SEM.
Discussion Recent reports that Peyer's patch derived Vicia villosa adherent cells secrete IL-5 (26) suggest that contrasuppression is in part mediated by the Th2 cells. However, to date, there has not been any evidence that a single molecule can enhance B cell responses in a system where Ts have been activated. The data presented in this paper show that at least two different molecular mechanisms distinct from IL-2, IL-4, IL-5, and IL-6 mediate contrasuppressive-like activity. Only TGF-/31 was sufficient to mediate this effect on its own. TGF-/31 is not responsible for all contrasuppressive activity since it differs from other CSFs. It is neutralized by specific anti-serum but the CSFs in the macrophage - T cell co-cultures are not. In addition, enhancement of IgG by CSFs can be neutralized with a specific mAb to Fc>R (2.4G2) but the effect of TGF-/3 cannot. Previous reports also show that contra-
suppression by other factors is isotype-specific and Igh-restricted (10,16,17,24,25). The source of the Igh restriction appears to be at the level of the Ig needed to interact with the co-culture supernatants since antigen presenting cells and T cells from Igh distinct animals produce functional CSFs as long as the Ig is of the identical Igh allotype as the source of the T cells (17). In contrast to the macrophage - T cell co-culture supernatants, TGF/31 did not have genetic restriction in the experiments reported here. Recent studies show that TGF-/31 can enhance isotype switch in LPS-stimulated B cells to produce IgA which is enhanced even more in the presence of IL-2 and IL-5 (22,23). This may be due to the ability of TGF-/3 to facilitate isotype switch (27,28). These observations suggest TGF-/3 may play a role in the intestine by
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PLUS ANTI-TGF-D Fig. 5. Kinetics of TGF-01 -mediated enhancement. TGF-01 (2 ng/ml) was added to suppressed cultures of BALB/c cells on different days (left-hand panel of A). This molecule only worked when added during the first 3 days of culture, suggesting it acted early or required several days to manifest its activity. When TGF-/J1 was added at the time the cultures were set, either directly to the cultures (right-hand panel of A) or incubated with the Ts (B) and then neutralized on different days (0 - 4) of a 5 day culture, its effect was rapidly neutralized at any time point tested. Results are expressed as the mean of nine replicates from three experiments ± SEM.
facilitating the generation of IgA responses. However, cytokines which work in the LPS-stimulated B cell system to enhance IgA, such as IL-5, do not account for the isotype-specific contrasuppression of IgA since they do not adhere to IgA (2) as seen
with the isotype-specific binding factors (Fig. 3) (16) found in the CSFs. Other cytokines which regulate antibody isotype of LPS-stimulated B cells were tested and did not, on their own, enhance responses in a suppressed system. TGF-/31 was only
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Fig. 6. TGF-/31 is not Igh-restricted. The CSFs in the supernatant from the macrophage-T cell co-cultures (sup) is present only when the cells and source of regulatory Ig are identical at the H2 and Igh locus with the responder cells in the read out system (cultures 3 and 4). Mixing co-culture supernatants made from the cells of one strain with the Ig from the Igh non-identical strain did not generate contrasuppression (cultures 5 and 6). However, TGF-/31 is not H2-restricted and does not require a source of exogenous Ig suggesting it is not Igh-restricted (culture 7). Results are expressed as the mean of three replicates from a representative experiment ± SEM.
active in suppressed cultures in the presence of antigen. These inconsistencies with respect to the effects of TGF-/3 on IgA may be explained by the differences between mitogen- and antigenstimulated systems. In other systems, TGF-/31 has been shown to down-regulate or inactivate many types of immune cells (29-31). Interestingly, TGF-/31 did not inhibit or enhance the T cell-dependent SRBC PFC response in control cultures nor did it alter the responses when responder cells were pulsed before culture suggesting it did not modulate antigen presenting cells, B cells or Th cells. The lack of inhibition may be due to our use of Marbrook - Diener cultures which allows low molecular weight toxins or inhibitory factors to escape into the larger media reservoir. This may be of significance in predicting the effects of TGF-/3 in vivo since blood flow would perfuse tissues and remove inhibitory substances induced by TGF-/3. The Th-derived isotype-specific binding factors have been shown to activate the Vicia w7/osa-adherent contrasuppressor T cells and not the Ts cell (L. Zettel and P. B. Ernst, in preparation). In contrast, incubation of the Ts with TGF-/31 abrogated the suppression while similar treatment of the responder cells had no effect. The type of Ts that was inhibited appears to be related to the isotype-specific suppressors for IgG, IgA, and IgE, whose mechanism of inhibition through FcR has been extensively studied (19,32-35). This suggests that we are studying the regulation of B cells that have already undergone isotype switch. The kinetics of TGF-/31 activity or its neutralization suggested
that the molecule required several days to confer its effect and it must be presented throughout the duration of the culture (Fig. 5A). Moreover, the effect of TGF-/31 on the Ts was rapidly reversible by the addition of specific antiserum after several days in culture. Together these observations suggest that TGF-/3 was carried over on a surface receptor on the Ts cell and that the addition of the antiserum competes for and removes the TGF-/3 which reverses its effect. The fact that most cells express receptors for TGF-/3 but the preincubation of the responders with TGF-/3 was insufficient to mediate the inhibitory effect further supports the hypothesis that the Ts was indeed the specific target. The rapidly reversible effect of anti-TGF-/3 on TGF-/3-treated Ts suggests that TGF-/3 affects the function of Ts and also rules out any cytotoxic effect as the mechanism. TGF-/31 has been shown to inhibit cytotoxic T cell activity mediated by CD8 + cells (36,37). Since TGF-/31 interferes with the suppression mediated by the CD8 + Ts added to our cultures, it is tempting to speculate that TGF-/31 may directly inhibit the suppressive and cytotoxic function of CD8 + cells. Based on reports that enhancement or inhibition of isotype-specific responses can be mediated through soluble binding factors differing only in their degree of glycosylation (38,39), TGF-/3 may modulate the production of these suppressive binding factors, perhaps by increasing their glycosylation. However, we were not able to find any evidence that antibodies to soluble Fc receptors/binding factors could interfere with the enhancement mediated by TGF-/3.
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Acknowledgements The authors wish to thank Drs R. Lynch, L. Mayer, and K. Croitoru for their comments and suggestions. This work was supported by grants from the Medical Research Council of Canada. D.A.C. is a recipient of an MRC Scientist award. Abbreviations CSF LPS PFC RES SRBC TGF-/31 Th Ts
contrasuppressive factors lipopolysaccharide plaque-forming cells responder cell population sheep red blood cell transforming growth factor-/31 helper T cells suppressor T cells
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