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T-dependent suppression of the primary antibody response to sheep erythrocytes in mice infected with Trichinella spiralis
CELLULAR
IMMUNOLOGY
27, 102-110
(1976)
T-Dependent Suppression of the Primary Antibody Response to Sheep Erythrocytes in M,ice Infected with Trichinella spiralis ’ JOYE
F.
Dcfiartment
JONES,
A.
CATHEKIXE
of Iwwnunology College of Medirixe,
CRANDALL,?
aud Medical University Received
AND
Microbiology Florida,
of
J&y
RICIIARD
aud ‘Department Gainesville, Florida
13.
CRANDALL
of
Pathology,
32610
6, 1976
Mice infected for 20 days with the parasitic nematode Trickinella spiralis had significantly reduced numbers of splenic antibody-forming cells (AFC) and decreased serum hemagglutinin titers following intraperitoneal immunization with sheep erythrocytes (SE). Similarly, when immunized i+a vitro to SE, cultures of splenocytes from infected mice developed fewer AFC than cultures of normal cells. Splenocytes from infected mice actively suppressed the in nitro response of normal cells to SE, and this in vitro suppression was abolished by lysis with anti-thy 1 antiserum and enhanced by lysis with anti-immunoglobulin antiserum. The addition of supernatant fluids from cultures of splenocytes from infected mice to cultures of normal cells on Day 0 of culture reduced by 70% the number of AFC produced by these cultures. These results indicate the presence of T-suppressor cells and suggest that antigen-induced supprexsion (antigenic competition) is one mechanism of Tricllilzclla-induced suppression.
INTRODUCTION Mice infected with the tissue-inhabiting nematode Trichinella spiralis have depressed antibody responses to sheep erythrocytes (SE) 3 (1, 2) and viruses (3) and delayed allograft rejection (4), although they have intense immune responses to parasite antigens (5). Similar suppression of immune responses to unrelated antigens has been reported in animals infected with a wide variety of other parasites (6-8). Although various hypotheses have been proposed to explain parasite-induced suppression, the mechanisms remain unclear. Since parasitic infections are prevalent throughout the world, an understanding of how parasitic infections induce altered states of inxnunologic req~onsiveness inay be important iii the ultimate control of many parasite infections. Therefore, we have investigated a cellular basis for tllis immunosuppression using a system of primary in vitro immunization. MATERIALdS
AXD
Alice. C57 E1/6J female mice (Jackson 10 weeks old were used in all experiments.
METHODS Laboratories,
Bar Harbor,
Me.)
6 to
1 Supported by Public Health Service Grant AI-03212 and Training Grant AI-00128-14 from NIAID. antigen-induced suppression ; Con 3 Abbreviations used : AFC, antibody-forming cells ; AIS, A, concanavalin A ; Ig, immunoglobulin ; LPS, lipopolysaccharide ; Z-ME, 2-mercaptoethanol ; PHA, phytohemagglutinin; SE, sheep erythrocytes; SEM, standard error of the mean; Tlymphocyte (T-cell), thymus-dependent lymphocyte. 102 Copyright All rights
0 1976 by Academic Press, of reproduction in any form
Inr. reserved.
SUPPRESSOR
T-CELLS
IN
T.
SI’IRALIS
103
INFECTION
Infection. Mice were infected orally with 200 Trichinella larvae by the method of Larsh and Kent (9). Sheep erythrorytes (SE). Sheep erythrocytes, obtained from a single sheep (Colorado Serum Co., Denver, Colo.), were washed three times in Dulbecco’s phosphate-buffered saline and stored up to 3 months in the medium described by Click et d. (10). Primwy in viva i~rfv~mixtior~. Groups of five normal and five infected mice were injected intraperitoneally with 0.2 ml of a 10% SE suspension. On Day 5, mice were exsanguinated and their spleens were removed and assayed for the numbers of direct antibody-forming cells (AFC). Three parasitized mice not receiving SE served as background controls; no AFC were detected in the spleens of these mice. Prinuq in eitro iml~1rt2i,-ation. Pouled spleens from five to twenty mice were cultured in zifro using the method of Click et 0.1.(10). T en million cells, to which 2 X 10’ SE were added, were cultured at 37°C in gas-tight boxes and were gassed daily with a mixture of 5% CO-, 12cjr OZ, and 8.3% X2. On Day 5, cultures were assayed for the number of direct AFC. Three to five replicates were cultured for each experimental group. Since cell recoveries were identical in all experimental and control groups within single experiments, data are presented as AFC per culture. Antibody-forwing r-ells (IZFC) . Direct (IgM) AFC Were enumerated by a slide modification ( 11) of the Jerne plaque assay ( 12). He?12agglzttinintifers. 2-Mercaptoethanol (2-ME)- sensitive (IgM) and -resistant (IgG) hemagglutinin titers were determined by microtiter techniques (13, 14). Antisera. Anti-thy 1.2 antiserum (donated by Dr. Bryan Gebharclt, Department of Pathology, University of Florida) was prepared in AKR mice by the injection of thymocytes from young CBA mice (15). Dilutions shown to kill at least 90% of a thymocyte suspensionwere used with rabbit complement to kill thy l-bearing splenocytes. Rabbit anti-mouse i~nt~~u~~oglobuli~~ (Ig) was prepared as described (16) and was shown by immunoelectrophoresis to recognize all mouse immunoglobulin classes.It was used with guinea pig complement at a concentration shown to kill 90% of splenic Tg-bearing cells. Mitogrn sfimztlnfion. Microtiter methods (17) were used to measure the proliferation of splenic lymphocytes in the presence of the mitogens concanavalin A (Con
TABLE Splenic
XI:C nays
liespouse postinfection
3 7 14 20
1
to Sheep Erythrocytes .~.-~-___--
Followillg
in
Percentage
of normal
Experiment
A
82 98 118 48”
Vko
Imn~unizatiw~
response’”
Experiment
B
72 89 93 536
‘I Meau of five individual mice. Percentage of normal response = [AFC mice) + AFC per spleen (normal mice)] X 100. * Iicductior~ in ~ZI~Cjsplcc~~ signilicxllt only nt I);ly 20. (I’ < 0.0.5).
per spleen
(infected
104
JONES,
CRANDALL
AND
CRANDALL
A ; Miles Labs, Inc., Kankakee, Ill.), phytohemagglutinin (PHA ; PHA-P, Difco Laboratories, Detroit, Mich.) , and Salmonella typhi+nurium lipopolysaccharide (LPS ; LPS-W, Difco). Cells were cultured for 72 hr with tritiated thymidine present during the last 24 hr of culture. Cultures were then harvested mechanically (Hiller Harvester, Otto Hiller Co., Madison, Wis.), and the number of counts present in an acid-insoluble product was measured. Four replicates were done of each culture. Collection of supernatant jluids. Splenocytes from normal or infected mice were cultured for 4 days in the absence of SE. Supernatant fluids were collected, centrifuged at 2OOg, and stored frozen until used. They were then concentrated lo-fold by negative-pressure dialysis and filtered through 0.22-pm Millipore filters. Statistics. Data were analyzed using Student’s t test, using log transformation of the AFC data. RESULTS In vivo inz~azlnosztppressio~l. Mice which had been infected with Trichinclla for varying lengths of time were injected with sheep erythrocytes (SE) and the number of splenic AFC and their serum hemagglutinin titers were determined. The data shown in Table 1 indicate that significant suppression of the AFC response in infected mice was not demonstrable until 20 days after infection. Likewise, the IgM serum hemagglutinin titers were suppressed in infected mice only on Day 20 (Table 2). A similar reduction was obtained in IgG titers (data not shown). Consequently, all further work reported here was carried out 21 -+ 1 days after infection. Splenic cell populations. To determine if there were differences in the cell populations of the spleens of infected and normal mice, differential cell counts and mitogen stimulations were done. As shown in Table 3, there was an increase in the proportion of large lymphocytes and blastoid cells and eosinophils with a ‘concomitant decrease in the proportion of small lymphocytes in the spleens of infected mice with no increase in total cell number. TABLE Serum
2
Hemagglutinin
Titers ____
Days postinfection
Mice
Log2 .___ Experiment
titer”
A
Experiment
B
3
Nb T
7.6 f 6.2 f
0.4 0.2
13.6 31 0.7 i
0.2
7
N
6.5 f 7.0 f
0.3 0.6
16.8 f 12.8 i
1.6 1.3
T
6.2 f 6.2 f
0.2 0.2
10.4 f 8.8 f
0.5
N T
9.6 f 7.2 f
0.4c 0.2
8.6 f 6.5 f
0.7c 0.5
T 14 20
N
0 Mean (f SEM) of five individual titers. * N = sera from normal mice; T = sera from c Titer of sera from infected mice significantly
Tvichinella-infected lower than normal.
11.8
mice.
1.1
SUPPRESSOR
T-CELLS
IN
Total
SPIRALIS
TABLE
3
Cell
Populations
Splenic Mice
‘I..
cells per spleen
Percentage Small lymphocytes
Normal Infected
*
8.8 x lo7 f 8.4 x 10’ f
0.6 X lo7 0.5 X lo7
79 f 60 f
of each cell type
Large lymphocytes”
0.7 4
105
INFECTION
16 zk 1 24 zt 3
a Also includes blastoid cells. * Normal values are the mean (& SEMI) of eight individual mice. of six individual mice infected 20 days previously with Trichinella.
Macrophages
Eosinophils
2 f 3fl
0 11 f4
Infected
0.3
values
are
the means
Splenocytes from normal and infected mice were stimulated in ztitro with the T-lymphocyte mitogens, Con A and PHA, and the B-lymphocyte mitogen, LPS. The data shown in Fig. 1 indicate that, while the LPS response of cells from infected mice was essentially normal, the responses to the T-lymphocyte mitogens were suppressed. In vitro i~~z~~~zf~zos~~pp~~cssioll. Normal splenocytes or those from mice infected 20 days previously with Trichinelln were cultured in the presence of SE for 5 days and the numbers of AFC were determined. Cultures of splenocytes from infected mice consistently developed fewer AFC than control cultures, e.g., in one typical experiment, splenocytes from normal mice produced 5500 f 450 AFC/culture 140 .
a
120 -
PHA ( p co.05
FIG. 1. Mitogen experiments within equals that of cells
LFprn, [cpn~,
CONA
1
l
LPS
(p<0.011 (p>oII
responses. Each point is one experiment; horizontal lines are means of each group. P value is probability that response of cells from infected mice from normal mice. Data are percentage of normal response: cells from ____infected cells front nornlal
mice (stimulated ~mice (stimulated
- background)] -
background)]
-
x 100.
106
JONES,
CRANDALL
AND
#--.-.-.-
-
I
__
_ -.
I
0
A
5.0
2.5 CELLS
FIG. 2. cells plus mice. (W) Mean of nificantly
CRANDALL
(x
ADDED
10-e)
Active suppression of normal AFC response k vitro. ( 0 ) Response of 10’ normal additional normal cells. (0) Response of 10’ normal cells plus cells from infected Response of lo7 cells from infected mice plus additional cells from infected mice. four replicate cultures (e SEM). Both doses of splenocytes from infected mice sigsuppressed the AFC response of normal cells (P < 0.05).
whereas only 900 + 80 AFC were obtained from splenocytes of infected mice. Similar results were obtained in eight other experiments. Since the immunosuppression could be “active,” due to the presence of a suppressive cell type, or “passive,” due to the lack of a particular cell type or alteration in the proportion of cells present, varying numbers of splenocytes from infected mice were added to cultures of 10 million normal cells. As shown in Fig. 2, both TABLE Effect
Experiment
of Anti-thy Activity
Treatment of added cells
4
and Anti-Ig Antisera on the Immunosuppressive of Splenocytes from Infected Mice AFC/culture* _-.
Number of added cells -Normal
A
None Anti-thy + C’
R
None Anti-Ig
+ C’
2.5 2.5
miceb
Infected
mice”
x x
106 106
8600 f 7870 f
800 480
3240 f 7460 f
280” 1000
1.25 x 1.25 x
106 106
6575 f 4630 f
1110 1140
5120 f 2630 f
230 460”
a Mean (* SEM) of four replicate cultures. * Source of added cells. c Signifmantly lower (P < 0.05) than other
three
vnlucs.
StrPI’RESSOR
Suppression
T-CELLS
IN
T.
SPIRALJS
of Normal AFC Response by Supernatant Fluids Cultures of Splenocytes from Infected Mice
Experiment
Percentage
=\ B C
l1 Percentage
reduction
cases, suppression b Day on which
Day
by Trichinella supernatant
1 -
(AFC/culture
with
2”
.~
71 9.5 75
=
from
reductiona
Day Ob ~___._.~._
_
107
INFECTIOS
56 57
not done T. spiralis
supernatant)
(AFC/culture with normal supernatants) supernatant was significant (I’ < 0.05). was added.
1
x ,Oo.
I
I ,
;,II
2.5 X 10” and 5 X 10c splenocytes from infected mice were able to suppress significantly the number of AFC produced by 10 million normal spleen cells, indicating active suppression due to the presence of a suppressor cell population. Similar suppression was observed in three other experiments. To investigate the identity of the suppressive population, splenocytes from normal or infected mice were treated with anti-thy 1 antiserum and complement ; 2.5 x 10” of the remaining viable cells were added to cultures of 10 x 10” normal cells. The results, shown in Table 4, Expt A, show that treatment of splenocytes with anti-thy Additionally, spleen cells from normal ant1 1 antiserum abolished suppression. infected mice were treated with anti-Ig plus complement, and 1.25 X 10” of the remaining cells were added to cultures of 10 X IO6 normal cells. As shown in Table 4, Expt P,, while these few cells from infected mice if untreatetl reduced only
FIG. 3. Addition of sera to normal cell cultures. ( l ) Sera from normal mice. (0) Sera from mice infected with Trichimlln 20 days previously. ( W) Fetal calf serum. Mean of four replicate cultures (t SEM).
108
JONES,
CRANDALL
AND
CRANDALL
slightly the normal response, treatment with anti-Tg, which enriches the T-cell population, significantly increased suppression. Iwwnunosuppressive factors. To determine if suppression was mediated through a soluble suppressor factor, supernatant fluids of cultures of splenocytes containing no SE were concentrated, and 0.6 ml (three culture-equivalents) was added to cultures of normal cells plus SE. As shown in Table 5, addition of supernatant fluids of cultures of cells from 20 day-infected mice significantly suppressed the response of normal splenocytes when added on either Day 0 or 2. Although supernatant fluids of normal cultures were suppressive, which has been shown by other workers (IS), supernatants from cultures of cells from infected mice were, significantly more suppressive. To determine if an immunosuppressive factor might be in the sera of infected mice, varying amounts of pooled sera from infected or normal mice were added to cultures of 10 million normal cells at the beginning of culture. The results, shown in Fig. 3, indicated that sera from infected mice were no more suppressive than sera from normal mice, and, since cell recoveries were identical in experimental and control cultures, the mouse sera were not cytotoxic. DISCUSSION Four important points have been established by the experiments presented in this paper. First, Trirlzinclla infection induces a suppression of the primary antiand this suppression can also be body response to SE 20 days after infection, demonstrated in cultures of splenocytes immunized in vitro. Second, splenocytes of infected mice, when added to cultures of normal cells, actively suppress the in vitro antibody response to SE. Third, this in vitro suppression is T-lymphocyte dependent since suppressor activity of the splenocytes is abolished by lysis with anti-thy 1 antiserum and is enhanced by lysis with anti-Ig antiserum. Fourth, supernatant fluids from cultures of splenocytes from infected mice suppress the primary in vitro antibody response of normal splenocytes. Immunosuppression induced by Triclzinelh infection has been extensively studied in mice, and suppression of the in vivo l~un~oral antibody response, as demonstrated in this study, is well established (1-4). Several hypotheses have been proposed to explain this immunosuppression. While there is no conclusive proof for any of these hypotheses, two proposed mechanisms of suppression have some experimental support : the presence of immunosuppressive (or toxic) factors derived from the helminth (2, 4, 19) and antigenic competition (20, 21). Faubert and Tanner have reported leukoagglutinating and leukotoxic factors in sera of Trichinella-infected mice, factors probably derived from the helminth, and have reported inhibition of the antibody response to SE by injection of such sera (19). Recently, Faubert reported that products of newborn larvae are suppressive for the primary antibody response to SE in vitro (21). Our in vitro studies or leukoagglutinating activity in sera from produced no evidence of leukotoxic infected mice since plaque responses and cell recoveries were identical in cultures containing sera from normal or infected mice. These experiments do not, however, exclude the possibility of hehninth-derived suppressor substances in Trichinella infection. One group of investigators has reported that the immune suppression induced by Triclainella infection is consistent with the described features of sequential antigenic
STTPT’RESSOK
T-CELLS
IX
‘I-.
SPTKALIS
~~FI’(“l-TON
109
cotiil~etitioii (I, LO). Antigenic competition, or antigen-intlucetl suppression (A 1S), is the suppression of an immune response to one antigen induced by the prior administration of another, unrelated antigen. The mechanisms of AIS are unclear (22), but it is induced by T-dependent lymphocytes stimulated during the initial immune response (23-25). Infection with Trichinella produces a complex, intense antigenic stimulation. At the time after infection that suppression is detected, mice are synthesizing large amounts of antibody, particularly IgG1, and their splenocytes are highly responsive to Trichinella antigens (5). The T-lymphocyte depenclencq of the immune responsesto Trichinclla infection has been demonstrated (26, 27). Thus, this helminth infection presents a situation in which antigenic competition could be active. Our results, which demonstrate T-suppressor cells, support the hypothesis that AIS contributes to the observed immunosuppression in Triclrinrlla infection. Several studies have demonstrated antigen-induced suppression ipz vitro which apparently are consistent with our ifz z~itro experimental results. \Yhen splenocytes from mice immunized with Trichinrlla in viva are cultured in ritro with SE, the antibody response to SE is impaired. This is similar to the demonstraton of AIS by SjGberg and Britton (25) and Pross et al. (28) who showed that the immune response to one antigen following in z&o immunization was suppressed if the splenocytes had been previously immunized in vizu to a different antigen. Additionally, Sjiiberg and Britton showed that splenocytes immunized in V~ZV to one antigen could suppress the antibody response of normal splenocytes immunized in z&o to another antigen (25), which parallels the evidence presented here of an active suppression by splenocytes from TrirIzinclla-infected mice. Furthermore, Thomas et al. showed that in vitro AIS is dependent on the production of a soluble suppressor factor (29), which is also consistent with our results. Although active immunosuppression induced by Triclzinclla infection has been demonstrated to be T-lymphocyte dependent, we cannot conclude that T-lymphocytes act directly to suppress antibody formation. The observed T-cell-dependent suppressioncould be mediated by another cell type normally present in the splenocyte population or by cells altered in number or function during Trichinella infection. Macrophages and T-lymphocytes have complex regulatory interactions (21 j and T-dependent, macrophage-mediated suppression has been demonstrated (30). Both suppressor T-lymphocytes (18), and macrophages (31) have been shown to suppressT-mitogen responses,and splenocytes of Trickinplla-infected mice have depressed PHA and Con A responses. The most notable change in splenocyte populations with Trirlzincllu infection is the increase in eosinophils. The possible role of eosinophils in immunosuppressionis unknown, but these cells are suggested to modulate allergic inflammatory responsesby synthesis and release of prostaglandins (32) and additional modulatory roles in antibody production cannot be excluded. Parasite infection often induces prominent T-dependent immmie responses,and one result may be the generation of suppressor T-lymphocytes and depression of immune responsiveness.For example, the experiments of Pelley et al. suggest that suppressor T-cells modulate the immunopathologic response in schistosomiasis (33). The significance of suppressor T-cells in the immune alterations associated with helminth infections requires further study, but our studies do suggest that AIS, dependent upon suppressor cells, may occur in several parasite infections and is probably important in altering the immune status of the host.
110
JONES,
CRANDALL
AND
CRAND.tLL
ACKNOWLEDGMENT We
thank
Charlene
Francis
for
her
expert
technical
assistance.
REFERENCES 1. Lubiniecki, A. S., and Cypess, R. H., Zlbfcrt. Zmrnrrn. 11, 1306, 197.5. 2. Faubert, G., and Tanner, C. E., Bxp. Parasitol. 380, 120, 1971. 3. Cypess, R. H., Lubiniecki, A. S., and Hammon, W. McD., Proc-. Sot. Exp. Biol. Med. 143, 469, 1973. 4. Svet-Moldavsky, G. J., Shaghijan, G. S., Chernyakhovskaya, I. Yu., Mkheidze, D. hl., Ozertskovskaya, N. N., and Kadaghidze, Z. G., Transplantat,ion 9, 69, 1970. 5. Crandall, R. B., and Crandall, C. A., Exg. Parasitol. 31, 378, 1972. 6. Strickland, G. T., Pettitt, L. E., and Voller, A., Amer. J. Trap. Mrd. Hyg. 22, 452, 1973. 7. Greenwood, B. M., In “Parasites in the Immunized Host: Mechanisms of Survival,” CIBR Foundation Symposium, pp. 137-159. Elsevier, New York, 1974. 8. Crandall, C. A., and Crandall, R. B., Exfi. Parasitol., in press. 9. Larsh, J. E., and Kent, D. E., J. Parasitol. 35, 45, 1949. 10. Click, R. E., Benck, L., and Alter, B. J., Cell. Zmmm~ol. 3, 264, 1972. 11. Mishell, R. I., and Dutton, R. W., J. Exp. Med. 126, 423, 1967. 12. Jerne, N. K., and Nordin, A. A., Science 140, 405, 1963. 13. Sever, J. L., /. Zmmrrnol. 8’8, 320, 1962. 14. Scott, D., and Gershon, R. K., Clin. Exp. Im~wnoI. 6, 313, 1970. 15. Cerottini, J. C., Nordin, A. A., and Brunner, K. T., Nahrc (Lmdorz) 228, 1308, 1970. 16. Crandall, C. A., and Crandall, R. B.. E.rp. I’artrsitol. 30. 426, 1972. 17. Hartzman, R. J., Bach, M. I.., Bach, F. H., Thurman, (;. I:., and Sell, K. W., Cc//. ZmmzuzoZ. 4, 182, 1972. 18. Kasahara, T., and Shioiri-Nakano, K., J. Z~rrnrrrtrol. 116, 1751, 1976. 19. Faubert, G. M., and Tanner, C. E., Z~r~rrrrr~olo~/~ 28, 1041, 1975. 20. Lubiniecki, A. S., Cypess, R. H., and Lucas, J. P., Tropmmcd. Pornsitol. 25, 345, 1974. 21. Faubert, G. M., Immunology 30, 485, 1976. 21. Liacopoulos, P., and Ben-Efraim, S., Proyv. AIlrrqy 18, 97, 1975. 23. Gershon, R. K., and Konda, K., J. Znrnrzr~roZ. 106, 1524, 1971. 24. Gershon, R. K., Conf~mp. Top. I~~rnu~~ohl. 3, 1, 1974. 25. Sjiiberg, O., and Britton, S., Eur. J. Zmmunol. 2, 282, 1972. 26. Dobson, C., In “Immunity to Animal Parasites” (E. J. L. Soulsby, Ed.), pp. 191-222. Academic Press, New York, 1972. 27. Perrudet-Badoux, A., Binaghi, R. A., and Biozzi, G., Zr~m~~moZogy 29, 387, 1975. 28. Pross, H., Novak, T., and Eidinger, D., Cell. ZmmzrnoE. 2, 44.5, 1971. 29. Thomas, D. W., Roberts, W. K., and Talmage, D. W., J. Inwztc~~ol. 114, 1616, 1975. 30. Gery, I., and Waksman, B. H., J. Exb. Med. 136, 143, 1972. 31. Kirchner, H., Chused, T. M., Herberman, R. B., Holden, H. T., and I,avrin, D. H., J. Exp. Med. 139, 1473, 1974. 32. Hubscher, T., J. Zmmunol. 114, 1389, 1975. 33. Pelley, R. P., Ruffier, J. J., and Warren, K. S., Znfrc-t. Z~~rrnrrz. 13, 1176, 1976.
IMMUNOLOGY
27, 102-110
(1976)
T-Dependent Suppression of the Primary Antibody Response to Sheep Erythrocytes in M,ice Infected with Trichinella spiralis ’ JOYE
F.
Dcfiartment
JONES,
A.
CATHEKIXE
of Iwwnunology College of Medirixe,
CRANDALL,?
aud Medical University Received
AND
Microbiology Florida,
of
J&y
RICIIARD
aud ‘Department Gainesville, Florida
13.
CRANDALL
of
Pathology,
32610
6, 1976
Mice infected for 20 days with the parasitic nematode Trickinella spiralis had significantly reduced numbers of splenic antibody-forming cells (AFC) and decreased serum hemagglutinin titers following intraperitoneal immunization with sheep erythrocytes (SE). Similarly, when immunized i+a vitro to SE, cultures of splenocytes from infected mice developed fewer AFC than cultures of normal cells. Splenocytes from infected mice actively suppressed the in nitro response of normal cells to SE, and this in vitro suppression was abolished by lysis with anti-thy 1 antiserum and enhanced by lysis with anti-immunoglobulin antiserum. The addition of supernatant fluids from cultures of splenocytes from infected mice to cultures of normal cells on Day 0 of culture reduced by 70% the number of AFC produced by these cultures. These results indicate the presence of T-suppressor cells and suggest that antigen-induced supprexsion (antigenic competition) is one mechanism of Tricllilzclla-induced suppression.
INTRODUCTION Mice infected with the tissue-inhabiting nematode Trichinella spiralis have depressed antibody responses to sheep erythrocytes (SE) 3 (1, 2) and viruses (3) and delayed allograft rejection (4), although they have intense immune responses to parasite antigens (5). Similar suppression of immune responses to unrelated antigens has been reported in animals infected with a wide variety of other parasites (6-8). Although various hypotheses have been proposed to explain parasite-induced suppression, the mechanisms remain unclear. Since parasitic infections are prevalent throughout the world, an understanding of how parasitic infections induce altered states of inxnunologic req~onsiveness inay be important iii the ultimate control of many parasite infections. Therefore, we have investigated a cellular basis for tllis immunosuppression using a system of primary in vitro immunization. MATERIALdS
AXD
Alice. C57 E1/6J female mice (Jackson 10 weeks old were used in all experiments.
METHODS Laboratories,
Bar Harbor,
Me.)
6 to
1 Supported by Public Health Service Grant AI-03212 and Training Grant AI-00128-14 from NIAID. antigen-induced suppression ; Con 3 Abbreviations used : AFC, antibody-forming cells ; AIS, A, concanavalin A ; Ig, immunoglobulin ; LPS, lipopolysaccharide ; Z-ME, 2-mercaptoethanol ; PHA, phytohemagglutinin; SE, sheep erythrocytes; SEM, standard error of the mean; Tlymphocyte (T-cell), thymus-dependent lymphocyte. 102 Copyright All rights
0 1976 by Academic Press, of reproduction in any form
Inr. reserved.
SUPPRESSOR
T-CELLS
IN
T.
SI’IRALIS
103
INFECTION
Infection. Mice were infected orally with 200 Trichinella larvae by the method of Larsh and Kent (9). Sheep erythrorytes (SE). Sheep erythrocytes, obtained from a single sheep (Colorado Serum Co., Denver, Colo.), were washed three times in Dulbecco’s phosphate-buffered saline and stored up to 3 months in the medium described by Click et d. (10). Primwy in viva i~rfv~mixtior~. Groups of five normal and five infected mice were injected intraperitoneally with 0.2 ml of a 10% SE suspension. On Day 5, mice were exsanguinated and their spleens were removed and assayed for the numbers of direct antibody-forming cells (AFC). Three parasitized mice not receiving SE served as background controls; no AFC were detected in the spleens of these mice. Prinuq in eitro iml~1rt2i,-ation. Pouled spleens from five to twenty mice were cultured in zifro using the method of Click et 0.1.(10). T en million cells, to which 2 X 10’ SE were added, were cultured at 37°C in gas-tight boxes and were gassed daily with a mixture of 5% CO-, 12cjr OZ, and 8.3% X2. On Day 5, cultures were assayed for the number of direct AFC. Three to five replicates were cultured for each experimental group. Since cell recoveries were identical in all experimental and control groups within single experiments, data are presented as AFC per culture. Antibody-forwing r-ells (IZFC) . Direct (IgM) AFC Were enumerated by a slide modification ( 11) of the Jerne plaque assay ( 12). He?12agglzttinintifers. 2-Mercaptoethanol (2-ME)- sensitive (IgM) and -resistant (IgG) hemagglutinin titers were determined by microtiter techniques (13, 14). Antisera. Anti-thy 1.2 antiserum (donated by Dr. Bryan Gebharclt, Department of Pathology, University of Florida) was prepared in AKR mice by the injection of thymocytes from young CBA mice (15). Dilutions shown to kill at least 90% of a thymocyte suspensionwere used with rabbit complement to kill thy l-bearing splenocytes. Rabbit anti-mouse i~nt~~u~~oglobuli~~ (Ig) was prepared as described (16) and was shown by immunoelectrophoresis to recognize all mouse immunoglobulin classes.It was used with guinea pig complement at a concentration shown to kill 90% of splenic Tg-bearing cells. Mitogrn sfimztlnfion. Microtiter methods (17) were used to measure the proliferation of splenic lymphocytes in the presence of the mitogens concanavalin A (Con
TABLE Splenic
XI:C nays
liespouse postinfection
3 7 14 20
1
to Sheep Erythrocytes .~.-~-___--
Followillg
in
Percentage
of normal
Experiment
A
82 98 118 48”
Vko
Imn~unizatiw~
response’”
Experiment
B
72 89 93 536
‘I Meau of five individual mice. Percentage of normal response = [AFC mice) + AFC per spleen (normal mice)] X 100. * Iicductior~ in ~ZI~Cjsplcc~~ signilicxllt only nt I);ly 20. (I’ < 0.0.5).
per spleen
(infected
104
JONES,
CRANDALL
AND
CRANDALL
A ; Miles Labs, Inc., Kankakee, Ill.), phytohemagglutinin (PHA ; PHA-P, Difco Laboratories, Detroit, Mich.) , and Salmonella typhi+nurium lipopolysaccharide (LPS ; LPS-W, Difco). Cells were cultured for 72 hr with tritiated thymidine present during the last 24 hr of culture. Cultures were then harvested mechanically (Hiller Harvester, Otto Hiller Co., Madison, Wis.), and the number of counts present in an acid-insoluble product was measured. Four replicates were done of each culture. Collection of supernatant jluids. Splenocytes from normal or infected mice were cultured for 4 days in the absence of SE. Supernatant fluids were collected, centrifuged at 2OOg, and stored frozen until used. They were then concentrated lo-fold by negative-pressure dialysis and filtered through 0.22-pm Millipore filters. Statistics. Data were analyzed using Student’s t test, using log transformation of the AFC data. RESULTS In vivo inz~azlnosztppressio~l. Mice which had been infected with Trichinclla for varying lengths of time were injected with sheep erythrocytes (SE) and the number of splenic AFC and their serum hemagglutinin titers were determined. The data shown in Table 1 indicate that significant suppression of the AFC response in infected mice was not demonstrable until 20 days after infection. Likewise, the IgM serum hemagglutinin titers were suppressed in infected mice only on Day 20 (Table 2). A similar reduction was obtained in IgG titers (data not shown). Consequently, all further work reported here was carried out 21 -+ 1 days after infection. Splenic cell populations. To determine if there were differences in the cell populations of the spleens of infected and normal mice, differential cell counts and mitogen stimulations were done. As shown in Table 3, there was an increase in the proportion of large lymphocytes and blastoid cells and eosinophils with a ‘concomitant decrease in the proportion of small lymphocytes in the spleens of infected mice with no increase in total cell number. TABLE Serum
2
Hemagglutinin
Titers ____
Days postinfection
Mice
Log2 .___ Experiment
titer”
A
Experiment
B
3
Nb T
7.6 f 6.2 f
0.4 0.2
13.6 31 0.7 i
0.2
7
N
6.5 f 7.0 f
0.3 0.6
16.8 f 12.8 i
1.6 1.3
T
6.2 f 6.2 f
0.2 0.2
10.4 f 8.8 f
0.5
N T
9.6 f 7.2 f
0.4c 0.2
8.6 f 6.5 f
0.7c 0.5
T 14 20
N
0 Mean (f SEM) of five individual titers. * N = sera from normal mice; T = sera from c Titer of sera from infected mice significantly
Tvichinella-infected lower than normal.
11.8
mice.
1.1
SUPPRESSOR
T-CELLS
IN
Total
SPIRALIS
TABLE
3
Cell
Populations
Splenic Mice
‘I..
cells per spleen
Percentage Small lymphocytes
Normal Infected
*
8.8 x lo7 f 8.4 x 10’ f
0.6 X lo7 0.5 X lo7
79 f 60 f
of each cell type
Large lymphocytes”
0.7 4
105
INFECTION
16 zk 1 24 zt 3
a Also includes blastoid cells. * Normal values are the mean (& SEMI) of eight individual mice. of six individual mice infected 20 days previously with Trichinella.
Macrophages
Eosinophils
2 f 3fl
0 11 f4
Infected
0.3
values
are
the means
Splenocytes from normal and infected mice were stimulated in ztitro with the T-lymphocyte mitogens, Con A and PHA, and the B-lymphocyte mitogen, LPS. The data shown in Fig. 1 indicate that, while the LPS response of cells from infected mice was essentially normal, the responses to the T-lymphocyte mitogens were suppressed. In vitro i~~z~~~zf~zos~~pp~~cssioll. Normal splenocytes or those from mice infected 20 days previously with Trichinelln were cultured in the presence of SE for 5 days and the numbers of AFC were determined. Cultures of splenocytes from infected mice consistently developed fewer AFC than control cultures, e.g., in one typical experiment, splenocytes from normal mice produced 5500 f 450 AFC/culture 140 .
a
120 -
PHA ( p co.05
FIG. 1. Mitogen experiments within equals that of cells
LFprn, [cpn~,
CONA
1
l
LPS
(p<0.011 (p>oII
responses. Each point is one experiment; horizontal lines are means of each group. P value is probability that response of cells from infected mice from normal mice. Data are percentage of normal response: cells from ____infected cells front nornlal
mice (stimulated ~mice (stimulated
- background)] -
background)]
-
x 100.
106
JONES,
CRANDALL
AND
#--.-.-.-
-
I
__
_ -.
I
0
A
5.0
2.5 CELLS
FIG. 2. cells plus mice. (W) Mean of nificantly
CRANDALL
(x
ADDED
10-e)
Active suppression of normal AFC response k vitro. ( 0 ) Response of 10’ normal additional normal cells. (0) Response of 10’ normal cells plus cells from infected Response of lo7 cells from infected mice plus additional cells from infected mice. four replicate cultures (e SEM). Both doses of splenocytes from infected mice sigsuppressed the AFC response of normal cells (P < 0.05).
whereas only 900 + 80 AFC were obtained from splenocytes of infected mice. Similar results were obtained in eight other experiments. Since the immunosuppression could be “active,” due to the presence of a suppressive cell type, or “passive,” due to the lack of a particular cell type or alteration in the proportion of cells present, varying numbers of splenocytes from infected mice were added to cultures of 10 million normal cells. As shown in Fig. 2, both TABLE Effect
Experiment
of Anti-thy Activity
Treatment of added cells
4
and Anti-Ig Antisera on the Immunosuppressive of Splenocytes from Infected Mice AFC/culture* _-.
Number of added cells -Normal
A
None Anti-thy + C’
R
None Anti-Ig
+ C’
2.5 2.5
miceb
Infected
mice”
x x
106 106
8600 f 7870 f
800 480
3240 f 7460 f
280” 1000
1.25 x 1.25 x
106 106
6575 f 4630 f
1110 1140
5120 f 2630 f
230 460”
a Mean (* SEM) of four replicate cultures. * Source of added cells. c Signifmantly lower (P < 0.05) than other
three
vnlucs.
StrPI’RESSOR
Suppression
T-CELLS
IN
T.
SPIRALJS
of Normal AFC Response by Supernatant Fluids Cultures of Splenocytes from Infected Mice
Experiment
Percentage
=\ B C
l1 Percentage
reduction
cases, suppression b Day on which
Day
by Trichinella supernatant
1 -
(AFC/culture
with
2”
.~
71 9.5 75
=
from
reductiona
Day Ob ~___._.~._
_
107
INFECTIOS
56 57
not done T. spiralis
supernatant)
(AFC/culture with normal supernatants) supernatant was significant (I’ < 0.05). was added.
1
x ,Oo.
I
I ,
;,II
2.5 X 10” and 5 X 10c splenocytes from infected mice were able to suppress significantly the number of AFC produced by 10 million normal spleen cells, indicating active suppression due to the presence of a suppressor cell population. Similar suppression was observed in three other experiments. To investigate the identity of the suppressive population, splenocytes from normal or infected mice were treated with anti-thy 1 antiserum and complement ; 2.5 x 10” of the remaining viable cells were added to cultures of 10 x 10” normal cells. The results, shown in Table 4, Expt A, show that treatment of splenocytes with anti-thy Additionally, spleen cells from normal ant1 1 antiserum abolished suppression. infected mice were treated with anti-Ig plus complement, and 1.25 X 10” of the remaining cells were added to cultures of 10 X IO6 normal cells. As shown in Table 4, Expt P,, while these few cells from infected mice if untreatetl reduced only
FIG. 3. Addition of sera to normal cell cultures. ( l ) Sera from normal mice. (0) Sera from mice infected with Trichimlln 20 days previously. ( W) Fetal calf serum. Mean of four replicate cultures (t SEM).
108
JONES,
CRANDALL
AND
CRANDALL
slightly the normal response, treatment with anti-Tg, which enriches the T-cell population, significantly increased suppression. Iwwnunosuppressive factors. To determine if suppression was mediated through a soluble suppressor factor, supernatant fluids of cultures of splenocytes containing no SE were concentrated, and 0.6 ml (three culture-equivalents) was added to cultures of normal cells plus SE. As shown in Table 5, addition of supernatant fluids of cultures of cells from 20 day-infected mice significantly suppressed the response of normal splenocytes when added on either Day 0 or 2. Although supernatant fluids of normal cultures were suppressive, which has been shown by other workers (IS), supernatants from cultures of cells from infected mice were, significantly more suppressive. To determine if an immunosuppressive factor might be in the sera of infected mice, varying amounts of pooled sera from infected or normal mice were added to cultures of 10 million normal cells at the beginning of culture. The results, shown in Fig. 3, indicated that sera from infected mice were no more suppressive than sera from normal mice, and, since cell recoveries were identical in experimental and control cultures, the mouse sera were not cytotoxic. DISCUSSION Four important points have been established by the experiments presented in this paper. First, Trirlzinclla infection induces a suppression of the primary antiand this suppression can also be body response to SE 20 days after infection, demonstrated in cultures of splenocytes immunized in vitro. Second, splenocytes of infected mice, when added to cultures of normal cells, actively suppress the in vitro antibody response to SE. Third, this in vitro suppression is T-lymphocyte dependent since suppressor activity of the splenocytes is abolished by lysis with anti-thy 1 antiserum and is enhanced by lysis with anti-Ig antiserum. Fourth, supernatant fluids from cultures of splenocytes from infected mice suppress the primary in vitro antibody response of normal splenocytes. Immunosuppression induced by Triclzinelh infection has been extensively studied in mice, and suppression of the in vivo l~un~oral antibody response, as demonstrated in this study, is well established (1-4). Several hypotheses have been proposed to explain this immunosuppression. While there is no conclusive proof for any of these hypotheses, two proposed mechanisms of suppression have some experimental support : the presence of immunosuppressive (or toxic) factors derived from the helminth (2, 4, 19) and antigenic competition (20, 21). Faubert and Tanner have reported leukoagglutinating and leukotoxic factors in sera of Trichinella-infected mice, factors probably derived from the helminth, and have reported inhibition of the antibody response to SE by injection of such sera (19). Recently, Faubert reported that products of newborn larvae are suppressive for the primary antibody response to SE in vitro (21). Our in vitro studies or leukoagglutinating activity in sera from produced no evidence of leukotoxic infected mice since plaque responses and cell recoveries were identical in cultures containing sera from normal or infected mice. These experiments do not, however, exclude the possibility of hehninth-derived suppressor substances in Trichinella infection. One group of investigators has reported that the immune suppression induced by Triclainella infection is consistent with the described features of sequential antigenic
STTPT’RESSOK
T-CELLS
IX
‘I-.
SPTKALIS
~~FI’(“l-TON
109
cotiil~etitioii (I, LO). Antigenic competition, or antigen-intlucetl suppression (A 1S), is the suppression of an immune response to one antigen induced by the prior administration of another, unrelated antigen. The mechanisms of AIS are unclear (22), but it is induced by T-dependent lymphocytes stimulated during the initial immune response (23-25). Infection with Trichinella produces a complex, intense antigenic stimulation. At the time after infection that suppression is detected, mice are synthesizing large amounts of antibody, particularly IgG1, and their splenocytes are highly responsive to Trichinella antigens (5). The T-lymphocyte depenclencq of the immune responsesto Trichinclla infection has been demonstrated (26, 27). Thus, this helminth infection presents a situation in which antigenic competition could be active. Our results, which demonstrate T-suppressor cells, support the hypothesis that AIS contributes to the observed immunosuppression in Triclrinrlla infection. Several studies have demonstrated antigen-induced suppression ipz vitro which apparently are consistent with our ifz z~itro experimental results. \Yhen splenocytes from mice immunized with Trichinrlla in viva are cultured in ritro with SE, the antibody response to SE is impaired. This is similar to the demonstraton of AIS by SjGberg and Britton (25) and Pross et al. (28) who showed that the immune response to one antigen following in z&o immunization was suppressed if the splenocytes had been previously immunized in vizu to a different antigen. Additionally, Sjiiberg and Britton showed that splenocytes immunized in V~ZV to one antigen could suppress the antibody response of normal splenocytes immunized in z&o to another antigen (25), which parallels the evidence presented here of an active suppression by splenocytes from TrirIzinclla-infected mice. Furthermore, Thomas et al. showed that in vitro AIS is dependent on the production of a soluble suppressor factor (29), which is also consistent with our results. Although active immunosuppression induced by Triclzinclla infection has been demonstrated to be T-lymphocyte dependent, we cannot conclude that T-lymphocytes act directly to suppress antibody formation. The observed T-cell-dependent suppressioncould be mediated by another cell type normally present in the splenocyte population or by cells altered in number or function during Trichinella infection. Macrophages and T-lymphocytes have complex regulatory interactions (21 j and T-dependent, macrophage-mediated suppression has been demonstrated (30). Both suppressor T-lymphocytes (18), and macrophages (31) have been shown to suppressT-mitogen responses,and splenocytes of Trickinplla-infected mice have depressed PHA and Con A responses. The most notable change in splenocyte populations with Trirlzincllu infection is the increase in eosinophils. The possible role of eosinophils in immunosuppressionis unknown, but these cells are suggested to modulate allergic inflammatory responsesby synthesis and release of prostaglandins (32) and additional modulatory roles in antibody production cannot be excluded. Parasite infection often induces prominent T-dependent immmie responses,and one result may be the generation of suppressor T-lymphocytes and depression of immune responsiveness.For example, the experiments of Pelley et al. suggest that suppressor T-cells modulate the immunopathologic response in schistosomiasis (33). The significance of suppressor T-cells in the immune alterations associated with helminth infections requires further study, but our studies do suggest that AIS, dependent upon suppressor cells, may occur in several parasite infections and is probably important in altering the immune status of the host.
110
JONES,
CRANDALL
AND
CRAND.tLL
ACKNOWLEDGMENT We
thank
Charlene
Francis
for
her
expert
technical
assistance.
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