- Email: [email protected]
Staphylococcus aureus-induced suppression of contact sensitivity in mice: Suppressor cells elicited by polyclonal B-cell activation are regulated by idiotype-anti-idiotype interactions
CELLULAR
IMMUNOLOGY
93, 508-5 19 (I 985)
Staphylococcus aureus-Induced Suppression of Contact Sensitivity in Mice: Suppressor Cells Elicited by Polyclonal B-Cell Activation Are Regulated by Idiotype-Anti-idiotype Interactions GIOIA BENEDETTINI,GENNARODE LIBERO, LUCIA MORI, PAOLA MARELLI, MARIA ROSARIA ANGIONI, AND MARIO CAMPA’ Institute of Microbiology,
University of Pisa, Via S. Zeno, 39, 56100 Pisa, Italy
Received January 8, 1985; accepted February 28, I985 Staphylococcus aureus strain Cowan I, a strong polyclonal B-cell activator (PBA), inhibited contact sensitivity to oxazolone in mice when administered 24 hr before sensitization. This suppression was mediated by idiotype-positive (Id+) B lymphocytes, which arose very early during the sensitization process and induced anti-Id B cells. These cells were found at Day 3 of the sensitization process and exerted their effect by activating antigen-specific suppressor T lymphocytes, which affected the efferent phase of the immune response. S. aureus strain Wood 46, which lacks of the ability to act as a PBA, was unable to inhibit contact sensitivity. These results indicate that PBA may play an important role in the regulation of cell-mediated immune reactions. 0 1985 Academic Press. Inc.
INTRODUCTION There is increasing evidence that B lymphocytes are involved in immunoregulation. It has been shown that Staphylococcus aureu~ induces suppressor B cells capable of inhibiting the onset of specific delayed-type hypersensitivity (DTH) to bacterial antigens (1). Pseudomonas aeruginosa as well as the lipopolysaccharide of gramnegative bacteria (LPS), and S. aureus strain Cowan I (StaCwI) affect contact sensitivity to oxazolone in mice, by activating antigen-specific suppressor B cells (2-6). B Lymphocytes capable of inhibiting cell-mediated immune reactions in an antigen nonspecific manner were observed in mice treated with killed cells of Cundida albicans (7). Similar findings were reported in individuals vaccinated with Mycobacterium bovis strain BCG (BCG) (8). Recent experiments in mice have also shown that the specific DTH to BCG is inhibited by anti-idiotype (anti-Id) antibodies (9), and that the suppression of contact sensitivity to oxazolone, following the administration of purified protein derivative from hf. tuberculosis (PPD), is mediated by both anti-antigen and anti-Id B lymphocytes ( 10). Since all these microorganisms and bacterial constituents can act as polyclonal B-cell activators (PBA) (5), the above observations indicate that PBA may play an important role in the regulation of cell-mediated immunity. i To whom correspondence should be sent. 508 0008-8749185$3.00 Copyright Q 1985 by Academic Press, Inc. All rig&ts of reproduction in any form reserved.
S. aureus-INDUCED SUPPRESSION OF CONTACT SENSITIVITY
509
As a model for the investigation of this interference, contact sensitivity to oxazolone was studied in mice that were injected 24 hr before sensitization with either of two strains of S. aureus which differ specifically in their ability to polyclonally activate B lymphocytes. Strain Cowan I is known to act as a strong PBA, whereas strain Wood 46 (StaWo) lacks of this ability (11). The results show that, unlike StaWo, StaCwI suppresses contact sensitivity to oxazolone by activating sequentially idiotype-positive and anti-idiotype anti-oxazolone B lymphocytes; these latter cells, in turn, appear to exert their effect by recruiting antigen-specific suppressor T lymphocytes. MATERIALS
AND METHODS
Animals. C57BL/6 mice from our breeding colony, 8-12 weeks old and sex matched, were used throughout this investigation. In each experiment the animals were allocated at random to the different groups. Each group consisted of 6 or 7 mice. Microorganisms. S. aureus strain Cowan I (with high content of protein A) and strain Wood 46 (with low content of protein A) (1 I), originally obtained from the National Collection of Type Cultures (London, U.K.) were used. The bacteria were killed as previously described (6). Mice were injected intravenously (iv) with killed bacterial cells corresponding to 1 X lo8 colony-forming units (CFU) in 0.5 ml of sterile saline. Sensitization and detection of contact sensitivity. Mice were sensitized by painting the skin of the abdomen and lower thorax with 0.2 ml of a 1.5% solution of 4-ethoxymethylene-2-phenyloxazolone (oxazolone; ox) or a 3% solution of picryl chloride (Pit) in absolute ethanol, both purchased from BDH (Poole, U.K). Sensitized mice were challenged 6 days later by painting their ears with a drop of 1% ox in olive oil. The quantification of contact sensitivity was made by measuring the increase in ear thickness with a micrometer 24 hr later (1 unit = 10e3cm). Ox and picrylsulfonic acid coupling to human serum albumin. Ox was coupled to human serum albumin (HSA) using the procedure described by Askenase and Asherson (12). HSA was conjugated with picrylsulfonic acid following the procedure described by Rittenberg and Amkraut (13). Anti-ox and anti-picryl chloride (anti-Pit) antibodies (Abs). Mice injected with StaCwI were sensitized 24 hr later with 0.2 ml of a solution of either 1.5% ox or 3% Pit. The animals were sacrificed 3 days after sensitization and the sera collected were used for purification of anti-ox and anti-Pit Abs. Both these Abs were purified by chromatography on antigen (oxazolonated or picrylated HSA, respectively) using the Sevatest Insolmer Kit (Sera-Lab, Crawley Down, Sussex, U.K.) in accordance with the procedure instructions. Anti-idiotype anti-ox and anti-Id anti-Pit Abs. Mice injected with StaCwI were sensitized 24 hr later with 0.2 ml of a solution of 1.5% ox or 3% Pit. Six days after sensitization these animals were sacrificed and the sera collected were used for the purification of anti-Id Abs by chromatography on antigen (anti-ox or anti-Pit Abs, respectively) following the procedure described above. Anti-Thy 1.2 serum treatment. In order to remove T lymphocytes, lymph node (LN) cells at a concentration of 20 X 106/ml were mixed with an equal volume of a monoclonal anti-mouse Thy 1.2 serum (New England Nuclear, Dreieich, F.R.G.)
510
BENEDETTINI
ET AL.
diluted 1:100 in Eagle’s medium and kept on ice for 45 min. After washing, the cells were mixed with an equal volume of fresh guinea pig complement diluted 1:5 in Eagle’s medium and incubated for 45 min at 37°C. This treatment killed 39-48% of the LN cells as judged by nigrosine dye exclusion. Separation of T and B lymphocytes. Cells from the draining LN, teased in Eagle’s medium supplemented with 10% fetal calf serum (FCS; Flow Laboratories, U.K.), were washed twice with the same medium and then filtered first through a glasswool column in order to remove macrophages ( 14), and subsequently through a nylon-wool column (15). As judged by immunofluorescence techniques, this method yielded: (i) effluent lymphocytes with 92-97% viability of which 87-96% were thetabearing cells; (ii) nylon-wool-adherent lymphocytes with 9 l-96% viability of which 4-8% bore theta antigen and 75-85% carried membrane surface immunoglobulins. This second fraction contained macrophages which were generally fewer than 2% of the total count. B-Cell separation. “Panning” experiments were performed following the method described by Wysocki and Sato (16) with some modifications. Briefly, tissue culture petri dishes (9-cm diameter) were coated with oxazolonated or picrylated HSA at 200 pg/ml or with purified anti-ox or anti-Pit Abs. After overnight incubation at 4°C the fluids were removed and saved for reuse. The plates were washed three times and neutralized with Eagle’s medium supplemented with 40% FCS for 30 min to limit nonspecific adherence. Panning was at 35 X lo6 live cells in 3.5 ml of Eagle’s medium supplemented with 10% FCS. The nylon-wool-enriched B cells were applied for 1 hr at 4°C to petri dishes coated with oxazolonated or picrylated HSA, or coated with purified anti-ox or anti-Pit Abs. After incubation, the plates were shaken and the cells poured ofi both the anti-ox-depleted B-cell subpopulation, obtained from the plates coated with oxazolonated HSA, and the anti-Id-depleted B-cell subpopulation, recovered from the plates coated with anti-ox Abs, corresponded to 45-50% of the original B cells. In order to recover the bound cells, the plates were filled with phosphate-buffered saline supplemented with 5% FCS, and the entire surface of each plate was flushed using a Pasteur pipet. Ox-bound B cells (anti-ox B-cell-enriched subpopulation), obtained from the plates coated with oxazolonated HSA, represented 16-21% of the original total, while anti-ox Absbound B cells (anti-Id B-cell-enriched subpopulation), recovered from the plates coated with anti-ox Abs, were 20-27%; Pit-bound B cells (anti-Pit B-cell-enriched subpopulation), obtained from the plates coated with picrylated HSA, corresponded to 12-16%, and anti-Pit Abs-bound B cells (anti-Id anti-Pit B-cell-enriched subpopulation), recovered from the plates coated with anti-Pit Abs, were 10-l 3% of the original total. T-Cell separation. Nylon-wool-effluent T lymphocytes were fractioned further by “panning” experiments as described above. Briefly, tissue petri dishes were coated with oxazolonated HSA at 200 pg/ml or with purified anti-Id anti-ox Abs. The cells recovered from the plates coated with oxazolonated HSA were: ox-binding (adherent) T cells (34% of the original total) and ox-nonbinding (nonadherent) T cells which represented 30% of the original T lymphocytes. From the plates coated with anti-Id Abs, the anti-Id binding (adherent) T cells represented 15% of the original T lymphocytes and the anti-Id nonbinding (nonadherent) T cells were 39% of the original total.
S. aureus-INDUCED
SUPPRESSION
OF CONTACT
SENSITIVITY
511
Statistical analysis. The data are expressedas means + standard errors. Student’s t test was used to compare differences between the means. RESULTS Characterization of the B lymphocytes involved in StaCwI-induced suppression. In previous studies it has been shown that the suppression of contact sensitivity to ox in mice given StaCwI 24 hr before sensitization is mediated by B lymphocytes (6). In order to characterize further these cells, the nylon-wool-enriched B cells, obtained from the draining LN of mice sensitized with ox 3 days earlier and injected with StaCwI 24 hr before sensitization, were separated by “panning” into four subpopulations: the anti-ox and the anti-Id B-cell-enriched or -depleted subpopulations. These cells were separately transferred iv into recipient mice sensitized with ox 1 hr earlier. The choice of this time interval between sensitization and cell transfer, and of this source of cells was made in view of the fact that in mice the passive transfer of contact sensitivity by draining LN cells peaks at Day 3 after sensitization (17). The results show that the anti-ox B cells inhibited contact sensitivity as did the anti-Id-depleted B cells, whereas the anti-ox-depleted B cells and the anti-Id B cells were ineffective (Fig. 1A). Anti-Pit B cells and anti-Id antiPit B cells were also transferred into recipient mice sensitized with ox and failed to affect contact sensitivity in these animals (data not shown). In order to investigate whether in StaCwI-injected mice the anti-Id B cells as well as the anti-ox B cells play a role at different times of the sensitization process, these cells, obtained from the same donors used in the first series of experiments, were transferred iv into recipient mice sensitized with ox either 3 or 6 days earlier. As shown in Fig. lB, while the anti-Id B lymphocytes were able to affect contact sensitivity in recipient mice sensitized with ox 3 days before cell transfer, the anti-ox B lymphocytes were not. Anti-Pit B cells and anti-Id anti-Pit B cells similarly failed to transfer suppression of contact sensitivity to ox (data not shown). It is interesting to notice that the anti-ox B cells were again capable of inhibiting contact sensitivity when transferred to ox-sensitized recipients at the moment of their challenge (Fig. 1C). Mice receiving StaWo before sensitization did not exhibit depression of contact sensitivity, even when StaWo was used at doses three times higher than that of StaCwI (data not shown). Characterization of the suppressor cells that inhibit the eflerent phase of contact sensitivity. In order to establish which cells were actually responsible for the suppression at the moment of challenge (i.e., at Day 6 after sensitization), B- and T-enriched subpopulations from mice sensitized with ox 6 days earlier and injected with StaCwI 24 hr before sensitization were transferred iv into recipient mice sensitized with ox 6 days earlier. The recipients were challenged 3 hr after cell transfer, and only those given the T cells exhibited a marked depression of contact sensitivity. The suppressive activity of the whole LN cell population was sensitive to treatment with anti-Thy 1.2 serum and complement (Fig. 2). Ox-sensitized recipients given LN cells from donors which had only been sensitized with ox 6 days earlier exhibited a normal response (data not shown). The suppression was antigen specific in that the suppressor T cells (Ts) from StaCwI-injected and ox-sensitized mice did not interfere with contact sensitivity of recipient mice sensitized with Pit (Fig. 3). The above results suggest that anti-Id B cells, already
512
BENEDETTINI
ET AL.
141 POSITIVE ANTI-OX
CONTROL
DEPLETED
B CELLS
ANTI-Id
B CELLS
DEPLETED
I<
B CELLS
ANTI-OX
ANTI-Id
I
1-j
P <
0.00,
I
l+
B CELLS
NEGATIVE
CONTROL
POSITIVE
CONTROL
P <
0.00;
fEl
ANTI-OX
DEPLETED
B CELLS
ANTI-OX
B CELLS
ANTI-Id
B CELLS
ANTI-Id
DEPLETED
I 1
h
P <
0.005
B CELLS
NEGATIVE
CONTROL
POSITIVE
CONTROL
T LY”PHOCYTES B LYMPHOCYTES ANTI-OX ANTI-OX
DEPLETED
NEGATIVE
P <
0.00;
B CELLS 8 CELLS
CONTROL
I 0
1
1 2
1
1
1
I
4
INCREASE
6
IN
1
I 6
EAR THICKNESS
I
I
I
I
10
(1O-3
I
I
12
cm)
FIG. 1. Contact sensitivity to ox in mice receiving various enriched LN cell populations (20 X 1O6cells) obtained from donors which had been sensitized with ox 3 days earlier and injected with StaCwI 24 hr before sensitization. Ox sensitization of recipients was performed at the time of (A), 3 days before (B), or 6 days before (C) cell transfer.
formed at Day 3 after sensitization, might induce the Ts that mediate the suppression at the moment of challenge. In order to test this hypothesis, anti-Id B cells, obtained from mice sensitized with ox 3 days earlier and also injected with StaCwI 24 hr before sensitization, were transferred iv into recipient mice 3 days after their sensitization. After 72 hr, the recipients were killed, the draining LN cells were separated into T- and B-enriched subpopulations, which were then transferred iv into mice sensitized with ox 6 days earlier. These animals were challenged with ox 3 hr after cell transfer and only mice given the T cells exhibited a significant depression of contact sensitivity. These data clearly indicate that anti-Id B cells do induce Ts (Fig. 4). Mice receiving anti-Id-depleted B cells did not possessany suppressive activity in their draining LN cell population (data not shown).
S. aureus-INDUCED SUPPRESSION OF CONTACT SENSITIVITY I) (1 L 0 K c
POSTTIVF
CO4TROL
513
KFCIPlt\l~
yr-
B LYYPtIOCYTt’ T
LYMPHOCYTtS
ANTI-THETA TREATED NkGA’PIVE
P <
0.005
+ C’ CELI
S
CONTROL
I”““’ 0 INCREASE
I 2
4 JN
FAR
‘THTCKNtSS
6
I
x (t”-3
cm)
FIG. 2. Contact sensitivity to ox in mice receiving (at the time of challenge) T or B lymphocytes (20 X 106) obtained from syngeneic donors which had been sensitized with ox 6 days earlier and injected with StaCwl 24 hr before sensitization.
In order to establish whether anti-Id B lymphocytes, in turn, might be induced by the Id+ anti-ox B lymphocytes, experiments were carried out following the experimental design described in Fig. 5. Anti-ox B cells obtained from mice sensitized with ox 3 days earlier and also injected with StaCwI 24 hr before sensitization, were transferred iv into recipient mice at the time of their sensitization. After 3 days these animals were sacrificed and the anti-Id B cells recovered were transferred iv into another group of recipients which had been sensitized with ox 3 days earlier. Six days after sensitization these recipients were killed, the draining LN cells were separated into T- and B-enriched subpopulations which were then transferred iv into recipient mice which had been sensitized with ox 6 days earlier. These animals were challenged with ox 3 hr after cell transfer and only mice given the T cells exhibited a significant depression of contact sensitivity. Ox-sensitized recipients, given anti-ox-depleted B cells at Day 0 or anti-Id-depleted B cells at Day 3, exhibited a normal response when challenged at Day 6 after sensitization: these findings indicate that these donors had no suppressor cells in their draining LN cell population (data not shown). In order to obtain further characterization, the Ts were separated by “panning” into four subpopulations: the ox-binding and the ox-nonbinding T cells and the anti-Id-binding and the anti-Id-nonbinding T lymphocytes. These four subpopulations
514
BENEDETTINI DONORS St.&w1
ET AL.
RECIPIENTS
injected 0
2; 2 42 cell I *
ox-5wbsit
irat
Opic-stn5itizat
POSITIVE T
6 .i c,
.ii
ion
challenge
ion
CONTROL
transfer
I
k
LYMPHOCYTES
NEGATlVE
I-J
CONTROL
INCREASE
IN
EAR
THICKNESS
(IO
-3
cm)
FIG. 3. Contact sensitivity to Pit in mice receiving (at the time of challenge) T lymphocytes (20 X 106)
obtained from donors which had been sensitized with ox 6 days earlier and injected with StaCwI 24 hr before sensitization.
were separately injected iv into recipient mice ( 15 X lo6 cells/mouse) at the moment of their challenge. The ox-binding as well as the anti-Id-binding T cells transferred suppression, whereas the ox-nonbinding as well as the anti-Id-nonbinding T lymphocytes did not, indicating that the T lymphocytes affecting the efferent phase of contact sensitivity were ox specific and Id+ (Fig. 6). It is interesting to notice that in these “panning” experiments F(ab’)* fragments derived from anti-Id Abs were equally capable of binding Ts (data not shown). Moreover, the nylon-wool-effluent T lymphocytes, obtained from the draining LN of mice sensitized with ox 6 days earlier and injected with StaCwI 24 hr before sensitization were similarly ox-specific and Id+ T cells (data not shown). Taken together, these data clearly indicate a sequential activation of Ts by antiId B lymphocytes and of these cells by Id+ anti-ox B lymphocytes. DISCUSSION These results show that StaCwI inhibits contact sensitivity to oxazolone by activating various suppressor cells: Id+ anti-ox B lymphocytes play a major role in the suppression of contact sensitivity very early after sensitization, anti-Id B lymphocytes are effective at Day 3 of the immunization process, and T lymphocytes are involved in the inhibition of the efferent phase of the response. The sequential
515
S. UW~WINDUCED SUPPRESSION OF CONTACT SENSITIVITY StaCwI
injected
*
ox-+t,nzit
POSITIVE
iL.3t
ion
CONTROL
B LYMPHOCYTES T
LYMPHOCYTES
NEGATIVE
Pi
0.001
CONTROL
I
““““‘I
0
2 INCREASE
4 IN
EAR
THICKNESS
x
b (10
-3
IO
cm)
4. Contact sensitivity to ox in mice receiving (at the time of challenge) T or B lymphocytes (20 106)obtained from donors sensitized with ox 6 days earlier and injected with anti-Id B cells (I5 X 106) 3 days after sensitization. Anti-Id B cells had been obtained from mice sensitized with ox 3 days earlier and injected with StaCwI 24 hr before sensitization. FIG.
X
appearance of these cells is due to the fact that they are integrated in a complex circuit. Ts, which represent the final pathway of the circuit, are activated by anti-Id B lymphocytes. The role of auto-anti-Id Abs in the regulation of contact sensitivity has already been studied extensively. Moorhead has shown that these Abs inhibit the efferent phase of contact sensitivity by activating a subset of T lymphocytes Ia+, Id+, which act in an antigen-nonspecific manner (18). By contrast, in the present study, the Ts are ox-binding and Id+, and they inhibit contact sensitivity specifically, since they failed to transfer suppression from ox-sensitized mice to recipients sensitized with Pit. Zembala et al. (19, 20) have shown that the T-suppressor circuit that affects contact sensitivity consists of two cell types: The first cell releases an antigen-specific soluble factor that induces the second cell to produce an antigennonspecific inhibitor when triggered by the antigen and major histocompatibility complex products. This latter cell resembles that described by Moorhead (18). Experiments are in progress to establish whether the Ts in our model actually exert their effect in an antigen-specific manner or resemble the first cell of the T-suppressor circuit described by Zembala et al. as acting by activating another subset of antigen-nonspecific Ts (19, 20). In the reports of Moorhead (18), and Sy et al. (2 l), the auto-anti-Id Abs involved in the regulation of contact sensitivity were described as occurring in the serum of
516
BENEDETTINI
POSITIVE
CONTROL
B LYMPHOCYTES
I
T LYMPHOCYTES
-+I
NEGATIVE
ET AL.
CONTROL
P<
0.001
I
I’
1
0
2 INCREASE
’
1 4
IN
EAR
““‘I 6 THICKNESS
6 (10e3
10 cm)
FIG. 5. Contact sensitivity to ox in mice receiving (at the time of challenge) T or B lymphocytes (20 X 106)obtained from donors sensitized with ox 6 days earlier and injected with anti-Id B cells (15 X 106) 3 days after sensitization. Anti-Id B cells had been obtained from mice sensitized with ox 3 days earlier and injected at the time of sensitization with anti-ox B cells (15 X 106).Anti-ox B cells, in turn, had been obtained from mice sensitized with ox 3 days earlier and injected with StaCwI 24 hr before sensitization.
mice 9- 15 days after sensitization. On the contrary, in the present study, anti-Id B lymphocytes could already be detected at Day 3 after sensitization. This early activation of anti-Id B cells could be due to the fact that in our model the animals received StaCwI, a PBA (1 l), before sensitization. A possible mechanism by which StaCwI can influence the early appearance of anti-Id B cells is that StaCwI brings about a nonspecific activation of B-cell clones, including the anti-Id B cells. The finding that LPS, another PBA, can directly induce anti-Id clones, lend support to this view (22). However, our results show that Id+ anti-ox B lymphocytes induce anti-Id B cells, although they do not exclude the possibility that these cells could have already been activated by StaCwI. Thus, the possibility exists that the generation of anti-Id B cells is the result of the StaCwI-induced polyclonal activation and the subsequent selection and expansion brought about by the Id+ anti-ox B lymphocytes. These cells could have been similarly formed in great numbers as a consequence of the combined activity of the PBA and the antigen: StaCwI could have induced a polyclonal activation of B-cell clones, and oxazolone, administered 24 hr later, could have selected and further expanded the antigen-specific B cells. This hypothesis is supported by the findings that the sera of StaCwI-injected and
517
S. aureus-INDUCED SUPPRESSION OF CONTACT SENSITIVITY St,aCwl
injected
POSTTIVI,
CONTROL
OX-NON-RINDING OX-RINDING
T CELLS
ANTI-Id
NON-BINDING
ANTI-Id
RINDLNC
NEGA’PIVE
/----
T CELLS T CELL<
I’ < o.ooj
I
T CTL1.S
CONTROL
FIG. 6. Contact sensitivity to ox in mice receiving (at the time of challenge) various enriched T-cell subpopulations (15 X lo6 cells) obtained from mice sensitized with ox 6 days earlier and injected with anti-Id B cells 3 days after sensitization. Anti-Id B cehs had been obtained from mice sensitized with ox 3 days earlier and injected with StaCwI 24 hr before sensitization.
ox-sensitized mice contain anti-ox Abs already at Day 3 after sensitization, whereas these Abs are not detectable in the sera of mice only sensitized or only injected with StaCwI (23). Therefore, it appears that the suppressive effect exerted by StaCwI +AFFERENT
PHASE+
EFFERENT PHASE
+
FIG. 7. Proposed mechanisms of StaCwl-induced suppression of contact sensitivity to oxazolone in mice. (+): activation; (-): suppression.
518
BENEDETTINI
ET AL.
is related to its ability to act as a PBA, and a possible scenario is that StaCwI contributes to a very early expansion of the Id+ anti-ox B cells, which promote, possibly in concert with StaCwI, the early appearance of the corresponding anti-Id B cells, which, in turn, lead to the formation of Ts. Moorhead has shown that Id+ T lymphocytes are the inducers of the circuit that regulates contact sensitivity in mice (18). The present results also demonstrate that Id+ B lymphocytes can take part in the regulation of contact sensitivity, provided that an adequate stimulus, such as a polyclonal B-cell activation, allow them to expand to a sufficient extent. In this context it is interesting to notice that, although in a completely different model, Id+ B lymphocytes have been recently shown to play a major role in activating Ts involved in the regulation of the antibody response to type III pneumococcal polysaccharide (24). The findings that anti-ox B lymphocytes inhibit contact sensitivity to ox when injected immediately before challenge, as well as before sensitization (see Fig. l), suggestthat they can be suppressive through other mechanisms, in addition to the induction of anti-Id B cells. Anti-ox B cells may combine directly, or through the production of Abs, with ox, and thus prevent it from reaching the specifically reactive T cells. This type of immunoregulation has been proposed for Abs by several authors (25-27) and would explain why the anti-ox B cells did not affect contact sensitivity in mice receiving these cells at Day 3 of the sensitization process, i.e., when the effector T cells are already formed (17). Moreover, the anti-ox B cells, by producing specific Abs, may lead to the formation of immune complexes, which are known to represent a further stimulus for the production of anti-Id Abs (28). These Abs, in turn, can trigger Ts (29, 30). Although the present results do not provide evidence whether the StaCwI-activated immunoregulatory circuit includes the same cells that arise during conventional sensitization or not, they strongly suggest that StaCwI, administered before the antigen, is capable of triggering a complex immunoregulatory circuit because of its property of PBA (Fig. 7). This view is further supported by the findings that mice receiving StaWo, which is a poor PBA if it is one at all (1 l), instead of StaCwI, before sensitization, did not exhibit depression of contact sensitivity. Taken together, these results may have relevant implications in view of the fact that almost all bacteria possessmitogenic properties for B lymphocytes and almost all B lymphocytes can be activated by PBA (31). Thus, in infections handled by cell-mediated immune processesthe possibility exists that parasite-derived B-cell mitogens drive the host’s immune response in favor of the invading microorganism already during the very early phases of infection by triggering an early activation of immunoregulatory circuits that affect cell-mediated immunity. The activation of these circuits, on the contrary, can be beneficial to the host in infections in which the antibody response plays a major role, such as those caused by S. aureus, whereas cell-mediated immune reactions may lead to tissue damage (1). ACKNOWLEDGMENTS This work was supported by grants from C.N.R. (Progetto finalizzato: controllo malattie da infezione) and Minister0 della P.I., Rome.
REFERENCES 1. Easmon, C. S. F., and Glynn, A. A., Immunology 38, 103, 1979. 2. Campa, M., Garzelli, C., Ferrannini, E., and Falcone, G., C/in. Exp. Immunol. 26, 355, 1976.
S. aureus-INDUCED SUPPRESSION OF CONTACT SENSITIVITY
519
3. Campa, M., Colizzi, V., Garzelli, C., Toca, L., and Falcone, G., Curr. Microbial. 4, 173, 1980. 4. Colizzi, V., and Bozzi, L., Ann. Immunol. (Paris) 13OC,659, 1979. 5. Campa, M., In “Bacterial and Viral inhibition and Modulation of Host Defences” (G. Falcone, M. Campa, H. Smith, and G. H. Scott, Eds.), pp. 135-157. Academic Press,London/New York, 1984. 6. Benedettini, G., De Libero, G., Mori, L., and Campa, M., Int. Arch. Allergy Appl. Immunol. 73, 269, 1984. 7. Rivas, V., and Rogers, T. J., J. Immunol. 130, 376, 1983. 8. Bona, C., Audibert, F., Juy, D., Chedid, L., C/in. Exp. Immunol. 26, 258, 1976. 9. Colizzi, V., Giuntini, M., Garzelli, C., Campa, M., and Falcone, G., Cell. Immunol. 80, 205, 1983. IO. Campa, M., Benedettini, G., De Libero, G., Mori, L., and Falcone, G., Infect. Immun. 45, 701, 1984. 1I. Waldman, T. A., and Broder, S., Adv. Immunol. 32, 1, 1982. 12. Askenase, P. W., and Asherson, G. L., Immunology 23, 289, 1972. 13. Rittenberg, M. B., and Amkraut, A. A., J. Immunol. 97, 421, 1966. 14. Julius, M. H., Simpson, E., and Herzenberg. L. A., Eur. J. Immunol. 3, 645, 1973. 15. Trizio, D., and Cudkowicz, G., J. Immunol. 113, 1093, 1974. 16. Wysocki, L. J., and Sato, V. L., Proc. Natl. Acad. Sci USA 75, 2844, 1978. 17. Asherson, G. L., and Zembala, M., Nature New Biol. 244, 176, 1973. 18. Moorhead, J. W., J. Exp. Med. 155, 820, 1982. 19. Zembala, M. A., Asherson, G. L., James, B. M. B., Stein, V. E., and Watkins, M. C., J. Immunol. 129, 1823, 1982.
20. 21. 22. 23. 24.
Zembala, M. A., Colizzi, V., Asherson, G. L., and Watkins, M. C., Immunology 52, 599, 1984. Sy, M. S., Moorhead, J. W., and Claman, H. N., J. Immunol. 123, 2593, 1979. Rose, M. L., Goldman, M., and Lambert, P. H., J. Immunol. 128, 2126, 1982. Falcone, G., Mori, L., Benedettini, G., De Libero, G., and Campa, M., Ig. Mod. 81, 729, 1984. Taylor, C. E., Stashak, P. W., Caldes, G., Prescott, B., Chused, T. E., Brooks, A., and Baker, P. J., J. Exp. Med. 158, 703, 1983.
25. Fitch, F. W., Prog. Allergy 19, 195, 1975. 26. Uhr, J. W., and Moller, G., Adv. Immunol. 8, 81, 1968. 27. Zubler, R. H., J. Immunol. 126, 557, 1981. 28. Klaus, G. G. B., Nature (London) 272, 265, 1978. 29. Sy, M. S., Bach, B. A., Dohi, Y., Nisonolf, A., Benacerraf, B., and Greene, M. I., J. Exp. Med. 150, 1216, 1979. 30. Yamamoto, H., Nonaka, M., and Katz, D. H., J. Exp. Med. 150, 818, 1979. 31. Falcone, G., and Campa, M., In “Microbial Perturbation of Host Defences” (F. O’Brady and H. Smith, Eds.), pp. 185-2 10. Academic Press,London/New York, 1981.
IMMUNOLOGY
93, 508-5 19 (I 985)
Staphylococcus aureus-Induced Suppression of Contact Sensitivity in Mice: Suppressor Cells Elicited by Polyclonal B-Cell Activation Are Regulated by Idiotype-Anti-idiotype Interactions GIOIA BENEDETTINI,GENNARODE LIBERO, LUCIA MORI, PAOLA MARELLI, MARIA ROSARIA ANGIONI, AND MARIO CAMPA’ Institute of Microbiology,
University of Pisa, Via S. Zeno, 39, 56100 Pisa, Italy
Received January 8, 1985; accepted February 28, I985 Staphylococcus aureus strain Cowan I, a strong polyclonal B-cell activator (PBA), inhibited contact sensitivity to oxazolone in mice when administered 24 hr before sensitization. This suppression was mediated by idiotype-positive (Id+) B lymphocytes, which arose very early during the sensitization process and induced anti-Id B cells. These cells were found at Day 3 of the sensitization process and exerted their effect by activating antigen-specific suppressor T lymphocytes, which affected the efferent phase of the immune response. S. aureus strain Wood 46, which lacks of the ability to act as a PBA, was unable to inhibit contact sensitivity. These results indicate that PBA may play an important role in the regulation of cell-mediated immune reactions. 0 1985 Academic Press. Inc.
INTRODUCTION There is increasing evidence that B lymphocytes are involved in immunoregulation. It has been shown that Staphylococcus aureu~ induces suppressor B cells capable of inhibiting the onset of specific delayed-type hypersensitivity (DTH) to bacterial antigens (1). Pseudomonas aeruginosa as well as the lipopolysaccharide of gramnegative bacteria (LPS), and S. aureus strain Cowan I (StaCwI) affect contact sensitivity to oxazolone in mice, by activating antigen-specific suppressor B cells (2-6). B Lymphocytes capable of inhibiting cell-mediated immune reactions in an antigen nonspecific manner were observed in mice treated with killed cells of Cundida albicans (7). Similar findings were reported in individuals vaccinated with Mycobacterium bovis strain BCG (BCG) (8). Recent experiments in mice have also shown that the specific DTH to BCG is inhibited by anti-idiotype (anti-Id) antibodies (9), and that the suppression of contact sensitivity to oxazolone, following the administration of purified protein derivative from hf. tuberculosis (PPD), is mediated by both anti-antigen and anti-Id B lymphocytes ( 10). Since all these microorganisms and bacterial constituents can act as polyclonal B-cell activators (PBA) (5), the above observations indicate that PBA may play an important role in the regulation of cell-mediated immunity. i To whom correspondence should be sent. 508 0008-8749185$3.00 Copyright Q 1985 by Academic Press, Inc. All rig&ts of reproduction in any form reserved.
S. aureus-INDUCED SUPPRESSION OF CONTACT SENSITIVITY
509
As a model for the investigation of this interference, contact sensitivity to oxazolone was studied in mice that were injected 24 hr before sensitization with either of two strains of S. aureus which differ specifically in their ability to polyclonally activate B lymphocytes. Strain Cowan I is known to act as a strong PBA, whereas strain Wood 46 (StaWo) lacks of this ability (11). The results show that, unlike StaWo, StaCwI suppresses contact sensitivity to oxazolone by activating sequentially idiotype-positive and anti-idiotype anti-oxazolone B lymphocytes; these latter cells, in turn, appear to exert their effect by recruiting antigen-specific suppressor T lymphocytes. MATERIALS
AND METHODS
Animals. C57BL/6 mice from our breeding colony, 8-12 weeks old and sex matched, were used throughout this investigation. In each experiment the animals were allocated at random to the different groups. Each group consisted of 6 or 7 mice. Microorganisms. S. aureus strain Cowan I (with high content of protein A) and strain Wood 46 (with low content of protein A) (1 I), originally obtained from the National Collection of Type Cultures (London, U.K.) were used. The bacteria were killed as previously described (6). Mice were injected intravenously (iv) with killed bacterial cells corresponding to 1 X lo8 colony-forming units (CFU) in 0.5 ml of sterile saline. Sensitization and detection of contact sensitivity. Mice were sensitized by painting the skin of the abdomen and lower thorax with 0.2 ml of a 1.5% solution of 4-ethoxymethylene-2-phenyloxazolone (oxazolone; ox) or a 3% solution of picryl chloride (Pit) in absolute ethanol, both purchased from BDH (Poole, U.K). Sensitized mice were challenged 6 days later by painting their ears with a drop of 1% ox in olive oil. The quantification of contact sensitivity was made by measuring the increase in ear thickness with a micrometer 24 hr later (1 unit = 10e3cm). Ox and picrylsulfonic acid coupling to human serum albumin. Ox was coupled to human serum albumin (HSA) using the procedure described by Askenase and Asherson (12). HSA was conjugated with picrylsulfonic acid following the procedure described by Rittenberg and Amkraut (13). Anti-ox and anti-picryl chloride (anti-Pit) antibodies (Abs). Mice injected with StaCwI were sensitized 24 hr later with 0.2 ml of a solution of either 1.5% ox or 3% Pit. The animals were sacrificed 3 days after sensitization and the sera collected were used for purification of anti-ox and anti-Pit Abs. Both these Abs were purified by chromatography on antigen (oxazolonated or picrylated HSA, respectively) using the Sevatest Insolmer Kit (Sera-Lab, Crawley Down, Sussex, U.K.) in accordance with the procedure instructions. Anti-idiotype anti-ox and anti-Id anti-Pit Abs. Mice injected with StaCwI were sensitized 24 hr later with 0.2 ml of a solution of 1.5% ox or 3% Pit. Six days after sensitization these animals were sacrificed and the sera collected were used for the purification of anti-Id Abs by chromatography on antigen (anti-ox or anti-Pit Abs, respectively) following the procedure described above. Anti-Thy 1.2 serum treatment. In order to remove T lymphocytes, lymph node (LN) cells at a concentration of 20 X 106/ml were mixed with an equal volume of a monoclonal anti-mouse Thy 1.2 serum (New England Nuclear, Dreieich, F.R.G.)
510
BENEDETTINI
ET AL.
diluted 1:100 in Eagle’s medium and kept on ice for 45 min. After washing, the cells were mixed with an equal volume of fresh guinea pig complement diluted 1:5 in Eagle’s medium and incubated for 45 min at 37°C. This treatment killed 39-48% of the LN cells as judged by nigrosine dye exclusion. Separation of T and B lymphocytes. Cells from the draining LN, teased in Eagle’s medium supplemented with 10% fetal calf serum (FCS; Flow Laboratories, U.K.), were washed twice with the same medium and then filtered first through a glasswool column in order to remove macrophages ( 14), and subsequently through a nylon-wool column (15). As judged by immunofluorescence techniques, this method yielded: (i) effluent lymphocytes with 92-97% viability of which 87-96% were thetabearing cells; (ii) nylon-wool-adherent lymphocytes with 9 l-96% viability of which 4-8% bore theta antigen and 75-85% carried membrane surface immunoglobulins. This second fraction contained macrophages which were generally fewer than 2% of the total count. B-Cell separation. “Panning” experiments were performed following the method described by Wysocki and Sato (16) with some modifications. Briefly, tissue culture petri dishes (9-cm diameter) were coated with oxazolonated or picrylated HSA at 200 pg/ml or with purified anti-ox or anti-Pit Abs. After overnight incubation at 4°C the fluids were removed and saved for reuse. The plates were washed three times and neutralized with Eagle’s medium supplemented with 40% FCS for 30 min to limit nonspecific adherence. Panning was at 35 X lo6 live cells in 3.5 ml of Eagle’s medium supplemented with 10% FCS. The nylon-wool-enriched B cells were applied for 1 hr at 4°C to petri dishes coated with oxazolonated or picrylated HSA, or coated with purified anti-ox or anti-Pit Abs. After incubation, the plates were shaken and the cells poured ofi both the anti-ox-depleted B-cell subpopulation, obtained from the plates coated with oxazolonated HSA, and the anti-Id-depleted B-cell subpopulation, recovered from the plates coated with anti-ox Abs, corresponded to 45-50% of the original B cells. In order to recover the bound cells, the plates were filled with phosphate-buffered saline supplemented with 5% FCS, and the entire surface of each plate was flushed using a Pasteur pipet. Ox-bound B cells (anti-ox B-cell-enriched subpopulation), obtained from the plates coated with oxazolonated HSA, represented 16-21% of the original total, while anti-ox Absbound B cells (anti-Id B-cell-enriched subpopulation), recovered from the plates coated with anti-ox Abs, were 20-27%; Pit-bound B cells (anti-Pit B-cell-enriched subpopulation), obtained from the plates coated with picrylated HSA, corresponded to 12-16%, and anti-Pit Abs-bound B cells (anti-Id anti-Pit B-cell-enriched subpopulation), recovered from the plates coated with anti-Pit Abs, were 10-l 3% of the original total. T-Cell separation. Nylon-wool-effluent T lymphocytes were fractioned further by “panning” experiments as described above. Briefly, tissue petri dishes were coated with oxazolonated HSA at 200 pg/ml or with purified anti-Id anti-ox Abs. The cells recovered from the plates coated with oxazolonated HSA were: ox-binding (adherent) T cells (34% of the original total) and ox-nonbinding (nonadherent) T cells which represented 30% of the original T lymphocytes. From the plates coated with anti-Id Abs, the anti-Id binding (adherent) T cells represented 15% of the original T lymphocytes and the anti-Id nonbinding (nonadherent) T cells were 39% of the original total.
S. aureus-INDUCED
SUPPRESSION
OF CONTACT
SENSITIVITY
511
Statistical analysis. The data are expressedas means + standard errors. Student’s t test was used to compare differences between the means. RESULTS Characterization of the B lymphocytes involved in StaCwI-induced suppression. In previous studies it has been shown that the suppression of contact sensitivity to ox in mice given StaCwI 24 hr before sensitization is mediated by B lymphocytes (6). In order to characterize further these cells, the nylon-wool-enriched B cells, obtained from the draining LN of mice sensitized with ox 3 days earlier and injected with StaCwI 24 hr before sensitization, were separated by “panning” into four subpopulations: the anti-ox and the anti-Id B-cell-enriched or -depleted subpopulations. These cells were separately transferred iv into recipient mice sensitized with ox 1 hr earlier. The choice of this time interval between sensitization and cell transfer, and of this source of cells was made in view of the fact that in mice the passive transfer of contact sensitivity by draining LN cells peaks at Day 3 after sensitization (17). The results show that the anti-ox B cells inhibited contact sensitivity as did the anti-Id-depleted B cells, whereas the anti-ox-depleted B cells and the anti-Id B cells were ineffective (Fig. 1A). Anti-Pit B cells and anti-Id antiPit B cells were also transferred into recipient mice sensitized with ox and failed to affect contact sensitivity in these animals (data not shown). In order to investigate whether in StaCwI-injected mice the anti-Id B cells as well as the anti-ox B cells play a role at different times of the sensitization process, these cells, obtained from the same donors used in the first series of experiments, were transferred iv into recipient mice sensitized with ox either 3 or 6 days earlier. As shown in Fig. lB, while the anti-Id B lymphocytes were able to affect contact sensitivity in recipient mice sensitized with ox 3 days before cell transfer, the anti-ox B lymphocytes were not. Anti-Pit B cells and anti-Id anti-Pit B cells similarly failed to transfer suppression of contact sensitivity to ox (data not shown). It is interesting to notice that the anti-ox B cells were again capable of inhibiting contact sensitivity when transferred to ox-sensitized recipients at the moment of their challenge (Fig. 1C). Mice receiving StaWo before sensitization did not exhibit depression of contact sensitivity, even when StaWo was used at doses three times higher than that of StaCwI (data not shown). Characterization of the suppressor cells that inhibit the eflerent phase of contact sensitivity. In order to establish which cells were actually responsible for the suppression at the moment of challenge (i.e., at Day 6 after sensitization), B- and T-enriched subpopulations from mice sensitized with ox 6 days earlier and injected with StaCwI 24 hr before sensitization were transferred iv into recipient mice sensitized with ox 6 days earlier. The recipients were challenged 3 hr after cell transfer, and only those given the T cells exhibited a marked depression of contact sensitivity. The suppressive activity of the whole LN cell population was sensitive to treatment with anti-Thy 1.2 serum and complement (Fig. 2). Ox-sensitized recipients given LN cells from donors which had only been sensitized with ox 6 days earlier exhibited a normal response (data not shown). The suppression was antigen specific in that the suppressor T cells (Ts) from StaCwI-injected and ox-sensitized mice did not interfere with contact sensitivity of recipient mice sensitized with Pit (Fig. 3). The above results suggest that anti-Id B cells, already
512
BENEDETTINI
ET AL.
141 POSITIVE ANTI-OX
CONTROL
DEPLETED
B CELLS
ANTI-Id
B CELLS
DEPLETED
I<
B CELLS
ANTI-OX
ANTI-Id
I
1-j
P <
0.00,
I
l+
B CELLS
NEGATIVE
CONTROL
POSITIVE
CONTROL
P <
0.00;
fEl
ANTI-OX
DEPLETED
B CELLS
ANTI-OX
B CELLS
ANTI-Id
B CELLS
ANTI-Id
DEPLETED
I 1
h
P <
0.005
B CELLS
NEGATIVE
CONTROL
POSITIVE
CONTROL
T LY”PHOCYTES B LYMPHOCYTES ANTI-OX ANTI-OX
DEPLETED
NEGATIVE
P <
0.00;
B CELLS 8 CELLS
CONTROL
I 0
1
1 2
1
1
1
I
4
INCREASE
6
IN
1
I 6
EAR THICKNESS
I
I
I
I
10
(1O-3
I
I
12
cm)
FIG. 1. Contact sensitivity to ox in mice receiving various enriched LN cell populations (20 X 1O6cells) obtained from donors which had been sensitized with ox 3 days earlier and injected with StaCwI 24 hr before sensitization. Ox sensitization of recipients was performed at the time of (A), 3 days before (B), or 6 days before (C) cell transfer.
formed at Day 3 after sensitization, might induce the Ts that mediate the suppression at the moment of challenge. In order to test this hypothesis, anti-Id B cells, obtained from mice sensitized with ox 3 days earlier and also injected with StaCwI 24 hr before sensitization, were transferred iv into recipient mice 3 days after their sensitization. After 72 hr, the recipients were killed, the draining LN cells were separated into T- and B-enriched subpopulations, which were then transferred iv into mice sensitized with ox 6 days earlier. These animals were challenged with ox 3 hr after cell transfer and only mice given the T cells exhibited a significant depression of contact sensitivity. These data clearly indicate that anti-Id B cells do induce Ts (Fig. 4). Mice receiving anti-Id-depleted B cells did not possessany suppressive activity in their draining LN cell population (data not shown).
S. aureus-INDUCED SUPPRESSION OF CONTACT SENSITIVITY I) (1 L 0 K c
POSTTIVF
CO4TROL
513
KFCIPlt\l~
yr-
B LYYPtIOCYTt’ T
LYMPHOCYTtS
ANTI-THETA TREATED NkGA’PIVE
P <
0.005
+ C’ CELI
S
CONTROL
I”““’ 0 INCREASE
I 2
4 JN
FAR
‘THTCKNtSS
6
I
x (t”-3
cm)
FIG. 2. Contact sensitivity to ox in mice receiving (at the time of challenge) T or B lymphocytes (20 X 106) obtained from syngeneic donors which had been sensitized with ox 6 days earlier and injected with StaCwl 24 hr before sensitization.
In order to establish whether anti-Id B lymphocytes, in turn, might be induced by the Id+ anti-ox B lymphocytes, experiments were carried out following the experimental design described in Fig. 5. Anti-ox B cells obtained from mice sensitized with ox 3 days earlier and also injected with StaCwI 24 hr before sensitization, were transferred iv into recipient mice at the time of their sensitization. After 3 days these animals were sacrificed and the anti-Id B cells recovered were transferred iv into another group of recipients which had been sensitized with ox 3 days earlier. Six days after sensitization these recipients were killed, the draining LN cells were separated into T- and B-enriched subpopulations which were then transferred iv into recipient mice which had been sensitized with ox 6 days earlier. These animals were challenged with ox 3 hr after cell transfer and only mice given the T cells exhibited a significant depression of contact sensitivity. Ox-sensitized recipients, given anti-ox-depleted B cells at Day 0 or anti-Id-depleted B cells at Day 3, exhibited a normal response when challenged at Day 6 after sensitization: these findings indicate that these donors had no suppressor cells in their draining LN cell population (data not shown). In order to obtain further characterization, the Ts were separated by “panning” into four subpopulations: the ox-binding and the ox-nonbinding T cells and the anti-Id-binding and the anti-Id-nonbinding T lymphocytes. These four subpopulations
514
BENEDETTINI DONORS St.&w1
ET AL.
RECIPIENTS
injected 0
2; 2 42 cell I *
ox-5wbsit
irat
Opic-stn5itizat
POSITIVE T
6 .i c,
.ii
ion
challenge
ion
CONTROL
transfer
I
k
LYMPHOCYTES
NEGATlVE
I-J
CONTROL
INCREASE
IN
EAR
THICKNESS
(IO
-3
cm)
FIG. 3. Contact sensitivity to Pit in mice receiving (at the time of challenge) T lymphocytes (20 X 106)
obtained from donors which had been sensitized with ox 6 days earlier and injected with StaCwI 24 hr before sensitization.
were separately injected iv into recipient mice ( 15 X lo6 cells/mouse) at the moment of their challenge. The ox-binding as well as the anti-Id-binding T cells transferred suppression, whereas the ox-nonbinding as well as the anti-Id-nonbinding T lymphocytes did not, indicating that the T lymphocytes affecting the efferent phase of contact sensitivity were ox specific and Id+ (Fig. 6). It is interesting to notice that in these “panning” experiments F(ab’)* fragments derived from anti-Id Abs were equally capable of binding Ts (data not shown). Moreover, the nylon-wool-effluent T lymphocytes, obtained from the draining LN of mice sensitized with ox 6 days earlier and injected with StaCwI 24 hr before sensitization were similarly ox-specific and Id+ T cells (data not shown). Taken together, these data clearly indicate a sequential activation of Ts by antiId B lymphocytes and of these cells by Id+ anti-ox B lymphocytes. DISCUSSION These results show that StaCwI inhibits contact sensitivity to oxazolone by activating various suppressor cells: Id+ anti-ox B lymphocytes play a major role in the suppression of contact sensitivity very early after sensitization, anti-Id B lymphocytes are effective at Day 3 of the immunization process, and T lymphocytes are involved in the inhibition of the efferent phase of the response. The sequential
515
S. UW~WINDUCED SUPPRESSION OF CONTACT SENSITIVITY StaCwI
injected
*
ox-+t,nzit
POSITIVE
iL.3t
ion
CONTROL
B LYMPHOCYTES T
LYMPHOCYTES
NEGATIVE
Pi
0.001
CONTROL
I
““““‘I
0
2 INCREASE
4 IN
EAR
THICKNESS
x
b (10
-3
IO
cm)
4. Contact sensitivity to ox in mice receiving (at the time of challenge) T or B lymphocytes (20 106)obtained from donors sensitized with ox 6 days earlier and injected with anti-Id B cells (I5 X 106) 3 days after sensitization. Anti-Id B cells had been obtained from mice sensitized with ox 3 days earlier and injected with StaCwI 24 hr before sensitization. FIG.
X
appearance of these cells is due to the fact that they are integrated in a complex circuit. Ts, which represent the final pathway of the circuit, are activated by anti-Id B lymphocytes. The role of auto-anti-Id Abs in the regulation of contact sensitivity has already been studied extensively. Moorhead has shown that these Abs inhibit the efferent phase of contact sensitivity by activating a subset of T lymphocytes Ia+, Id+, which act in an antigen-nonspecific manner (18). By contrast, in the present study, the Ts are ox-binding and Id+, and they inhibit contact sensitivity specifically, since they failed to transfer suppression from ox-sensitized mice to recipients sensitized with Pit. Zembala et al. (19, 20) have shown that the T-suppressor circuit that affects contact sensitivity consists of two cell types: The first cell releases an antigen-specific soluble factor that induces the second cell to produce an antigennonspecific inhibitor when triggered by the antigen and major histocompatibility complex products. This latter cell resembles that described by Moorhead (18). Experiments are in progress to establish whether the Ts in our model actually exert their effect in an antigen-specific manner or resemble the first cell of the T-suppressor circuit described by Zembala et al. as acting by activating another subset of antigen-nonspecific Ts (19, 20). In the reports of Moorhead (18), and Sy et al. (2 l), the auto-anti-Id Abs involved in the regulation of contact sensitivity were described as occurring in the serum of
516
BENEDETTINI
POSITIVE
CONTROL
B LYMPHOCYTES
I
T LYMPHOCYTES
-+I
NEGATIVE
ET AL.
CONTROL
P<
0.001
I
I’
1
0
2 INCREASE
’
1 4
IN
EAR
““‘I 6 THICKNESS
6 (10e3
10 cm)
FIG. 5. Contact sensitivity to ox in mice receiving (at the time of challenge) T or B lymphocytes (20 X 106)obtained from donors sensitized with ox 6 days earlier and injected with anti-Id B cells (15 X 106) 3 days after sensitization. Anti-Id B cells had been obtained from mice sensitized with ox 3 days earlier and injected at the time of sensitization with anti-ox B cells (15 X 106).Anti-ox B cells, in turn, had been obtained from mice sensitized with ox 3 days earlier and injected with StaCwI 24 hr before sensitization.
mice 9- 15 days after sensitization. On the contrary, in the present study, anti-Id B lymphocytes could already be detected at Day 3 after sensitization. This early activation of anti-Id B cells could be due to the fact that in our model the animals received StaCwI, a PBA (1 l), before sensitization. A possible mechanism by which StaCwI can influence the early appearance of anti-Id B cells is that StaCwI brings about a nonspecific activation of B-cell clones, including the anti-Id B cells. The finding that LPS, another PBA, can directly induce anti-Id clones, lend support to this view (22). However, our results show that Id+ anti-ox B lymphocytes induce anti-Id B cells, although they do not exclude the possibility that these cells could have already been activated by StaCwI. Thus, the possibility exists that the generation of anti-Id B cells is the result of the StaCwI-induced polyclonal activation and the subsequent selection and expansion brought about by the Id+ anti-ox B lymphocytes. These cells could have been similarly formed in great numbers as a consequence of the combined activity of the PBA and the antigen: StaCwI could have induced a polyclonal activation of B-cell clones, and oxazolone, administered 24 hr later, could have selected and further expanded the antigen-specific B cells. This hypothesis is supported by the findings that the sera of StaCwI-injected and
517
S. aureus-INDUCED SUPPRESSION OF CONTACT SENSITIVITY St,aCwl
injected
POSTTIVI,
CONTROL
OX-NON-RINDING OX-RINDING
T CELLS
ANTI-Id
NON-BINDING
ANTI-Id
RINDLNC
NEGA’PIVE
/----
T CELLS T CELL<
I’ < o.ooj
I
T CTL1.S
CONTROL
FIG. 6. Contact sensitivity to ox in mice receiving (at the time of challenge) various enriched T-cell subpopulations (15 X lo6 cells) obtained from mice sensitized with ox 6 days earlier and injected with anti-Id B cells 3 days after sensitization. Anti-Id B cehs had been obtained from mice sensitized with ox 3 days earlier and injected with StaCwI 24 hr before sensitization.
ox-sensitized mice contain anti-ox Abs already at Day 3 after sensitization, whereas these Abs are not detectable in the sera of mice only sensitized or only injected with StaCwI (23). Therefore, it appears that the suppressive effect exerted by StaCwI +AFFERENT
PHASE+
EFFERENT PHASE
+
FIG. 7. Proposed mechanisms of StaCwl-induced suppression of contact sensitivity to oxazolone in mice. (+): activation; (-): suppression.
518
BENEDETTINI
ET AL.
is related to its ability to act as a PBA, and a possible scenario is that StaCwI contributes to a very early expansion of the Id+ anti-ox B cells, which promote, possibly in concert with StaCwI, the early appearance of the corresponding anti-Id B cells, which, in turn, lead to the formation of Ts. Moorhead has shown that Id+ T lymphocytes are the inducers of the circuit that regulates contact sensitivity in mice (18). The present results also demonstrate that Id+ B lymphocytes can take part in the regulation of contact sensitivity, provided that an adequate stimulus, such as a polyclonal B-cell activation, allow them to expand to a sufficient extent. In this context it is interesting to notice that, although in a completely different model, Id+ B lymphocytes have been recently shown to play a major role in activating Ts involved in the regulation of the antibody response to type III pneumococcal polysaccharide (24). The findings that anti-ox B lymphocytes inhibit contact sensitivity to ox when injected immediately before challenge, as well as before sensitization (see Fig. l), suggestthat they can be suppressive through other mechanisms, in addition to the induction of anti-Id B cells. Anti-ox B cells may combine directly, or through the production of Abs, with ox, and thus prevent it from reaching the specifically reactive T cells. This type of immunoregulation has been proposed for Abs by several authors (25-27) and would explain why the anti-ox B cells did not affect contact sensitivity in mice receiving these cells at Day 3 of the sensitization process, i.e., when the effector T cells are already formed (17). Moreover, the anti-ox B cells, by producing specific Abs, may lead to the formation of immune complexes, which are known to represent a further stimulus for the production of anti-Id Abs (28). These Abs, in turn, can trigger Ts (29, 30). Although the present results do not provide evidence whether the StaCwI-activated immunoregulatory circuit includes the same cells that arise during conventional sensitization or not, they strongly suggest that StaCwI, administered before the antigen, is capable of triggering a complex immunoregulatory circuit because of its property of PBA (Fig. 7). This view is further supported by the findings that mice receiving StaWo, which is a poor PBA if it is one at all (1 l), instead of StaCwI, before sensitization, did not exhibit depression of contact sensitivity. Taken together, these results may have relevant implications in view of the fact that almost all bacteria possessmitogenic properties for B lymphocytes and almost all B lymphocytes can be activated by PBA (31). Thus, in infections handled by cell-mediated immune processesthe possibility exists that parasite-derived B-cell mitogens drive the host’s immune response in favor of the invading microorganism already during the very early phases of infection by triggering an early activation of immunoregulatory circuits that affect cell-mediated immunity. The activation of these circuits, on the contrary, can be beneficial to the host in infections in which the antibody response plays a major role, such as those caused by S. aureus, whereas cell-mediated immune reactions may lead to tissue damage (1). ACKNOWLEDGMENTS This work was supported by grants from C.N.R. (Progetto finalizzato: controllo malattie da infezione) and Minister0 della P.I., Rome.
REFERENCES 1. Easmon, C. S. F., and Glynn, A. A., Immunology 38, 103, 1979. 2. Campa, M., Garzelli, C., Ferrannini, E., and Falcone, G., C/in. Exp. Immunol. 26, 355, 1976.
S. aureus-INDUCED SUPPRESSION OF CONTACT SENSITIVITY
519
3. Campa, M., Colizzi, V., Garzelli, C., Toca, L., and Falcone, G., Curr. Microbial. 4, 173, 1980. 4. Colizzi, V., and Bozzi, L., Ann. Immunol. (Paris) 13OC,659, 1979. 5. Campa, M., In “Bacterial and Viral inhibition and Modulation of Host Defences” (G. Falcone, M. Campa, H. Smith, and G. H. Scott, Eds.), pp. 135-157. Academic Press,London/New York, 1984. 6. Benedettini, G., De Libero, G., Mori, L., and Campa, M., Int. Arch. Allergy Appl. Immunol. 73, 269, 1984. 7. Rivas, V., and Rogers, T. J., J. Immunol. 130, 376, 1983. 8. Bona, C., Audibert, F., Juy, D., Chedid, L., C/in. Exp. Immunol. 26, 258, 1976. 9. Colizzi, V., Giuntini, M., Garzelli, C., Campa, M., and Falcone, G., Cell. Immunol. 80, 205, 1983. IO. Campa, M., Benedettini, G., De Libero, G., Mori, L., and Falcone, G., Infect. Immun. 45, 701, 1984. 1I. Waldman, T. A., and Broder, S., Adv. Immunol. 32, 1, 1982. 12. Askenase, P. W., and Asherson, G. L., Immunology 23, 289, 1972. 13. Rittenberg, M. B., and Amkraut, A. A., J. Immunol. 97, 421, 1966. 14. Julius, M. H., Simpson, E., and Herzenberg. L. A., Eur. J. Immunol. 3, 645, 1973. 15. Trizio, D., and Cudkowicz, G., J. Immunol. 113, 1093, 1974. 16. Wysocki, L. J., and Sato, V. L., Proc. Natl. Acad. Sci USA 75, 2844, 1978. 17. Asherson, G. L., and Zembala, M., Nature New Biol. 244, 176, 1973. 18. Moorhead, J. W., J. Exp. Med. 155, 820, 1982. 19. Zembala, M. A., Asherson, G. L., James, B. M. B., Stein, V. E., and Watkins, M. C., J. Immunol. 129, 1823, 1982.
20. 21. 22. 23. 24.
Zembala, M. A., Colizzi, V., Asherson, G. L., and Watkins, M. C., Immunology 52, 599, 1984. Sy, M. S., Moorhead, J. W., and Claman, H. N., J. Immunol. 123, 2593, 1979. Rose, M. L., Goldman, M., and Lambert, P. H., J. Immunol. 128, 2126, 1982. Falcone, G., Mori, L., Benedettini, G., De Libero, G., and Campa, M., Ig. Mod. 81, 729, 1984. Taylor, C. E., Stashak, P. W., Caldes, G., Prescott, B., Chused, T. E., Brooks, A., and Baker, P. J., J. Exp. Med. 158, 703, 1983.
25. Fitch, F. W., Prog. Allergy 19, 195, 1975. 26. Uhr, J. W., and Moller, G., Adv. Immunol. 8, 81, 1968. 27. Zubler, R. H., J. Immunol. 126, 557, 1981. 28. Klaus, G. G. B., Nature (London) 272, 265, 1978. 29. Sy, M. S., Bach, B. A., Dohi, Y., Nisonolf, A., Benacerraf, B., and Greene, M. I., J. Exp. Med. 150, 1216, 1979. 30. Yamamoto, H., Nonaka, M., and Katz, D. H., J. Exp. Med. 150, 818, 1979. 31. Falcone, G., and Campa, M., In “Microbial Perturbation of Host Defences” (F. O’Brady and H. Smith, Eds.), pp. 185-2 10. Academic Press,London/New York, 1981.