63, 21-31 (1987)
Nematospiroides dubius and Nippostrongylus Type Hypersensitivity Responses to Ovalbumin
brasiliensis: Delayed in the Infected Mouse
PATRICIA PRICE AND K. J. TURNER Clinicul
Unit. Princess Margaret (Accepted
13 June 1986)
PRICE, P., AND TURNER, K. J. 1987. Nematospiroides dabius and Nippostrongylus brasiliensis: Delayed type hypersensitivity responses to ovalbumin in the infected mouse. Experimental Parasitology 63, 21-31. Mice (C57BL) infected with the intestinal nematode Nematospiroides dubius showed depressed delayed type hypersensitivity responses to ovalbumin administered subcutaneously in Freund’s complete adjuvant. IgG and IgM responses to this inoculum were unaffected. It is unlikely that the depression arose from impairment of the ear test response because responses to an extract of the adult parasite were measurable and ear testing with lipopolysaccharide yielded normal responses in infected mice. Furthermore, mice immunized on the day of infection responded normally, whilst long term infected mice ear challenged with antigen pulsed macrophages gave depressed responses. The in vitro proliferative responses of cells from the spleens and from the lymph nodes draining the site of immunization were enhanced marginally by N. dub& infection. Furthermore, these cells induced normal or elevated adoptive delayed-type hypersensitivity and IgG responses in irradiated recipients. These findings suggest that N. dubius does not compromise the development of ovalbumin specific T cells involved in a delayed type hypersensitivity response. Evidence for the induction of suppressor cells by N. dabius is discussed, and the findings are compared with results obtained with Nipposrrongylas brasiliensis, a parasite which is rejected rapidly from the mouse. 0 1987 Academic Presr. Inc. INDEX DESCRIPTORS AND ABBREVIATIONS: Nernotospiroides dubias; Nippostrongylus brasiliensis; Nematodes, parasitic; Immunosuppression, not specific; Delayed type hypersensitivity (DTH); T-suppressor cells; Purified protein derivative (PPD); Phosphate buffered saline (PBS); Ovalbumin (OA); Freund’s complete adjuvant (FCA); Freund’s incomplete adjuvant (FIA); Lipopolysaccharide (LPS).
While broad spectrum immunosuppression has often been proposed as a factor contributing to the survival of a number of parasites, there have never been satisfactory answers to the basic questions of how much of the immune system is affected, how suppression is achieved, and whether it influences (or even correlates with) parasite survival. Argueably, the least well studied parasites in this regard are the intestinal helminths, for which the long lived murine worm, Nematospiroides dub&, is a workable model. To date, our experi-
ments have shown that N. dubius suppresses IgG responses to ovalbumin administered intraperitoneally with aluminium hydroxide during its encystment phase and that this involves the generation of immunosuppressive macrophages within the peritoneal cavity. There was no evidence of a role for suppressor T cells in this phenomenon, and the macrophage mediated effect declined once the worms were in the lumen of the intestine (Price and Turner 1984; Price and Holt 1986). Studies on the effects of immunosuppressive steroids on infections with Trichuris muris (Lee and Wakelin 1982) have shown that
0014-4894/87 $3.00 Copyright 0 1987 hy Academic Press. Inc All rights of reproduction in any form reserved.
parasites which can be made to survive the first week of infection (by cortisone treatment or perhaps via the generation of suppressive macrophages) will persist indefinitely protected by a state akin to “immunological tolerance.” Suppressor T cells are logical contenders for this role and their existence in sheep infected with Haemonchus contortus has already been postulated (Adams and Davies 1982). This parasite is totally gut dwelling like N. dubius, but similar conclusions have been reached with a Leishmania sp. (Howard et al. 1980) and a Schistosomas sp. (Pelley et al. 1976). The aims of the present experiments were to establish whether helminthic parasites can suppress cell mediated immunity and to investigate the mechanisms involved and possible correlations with parasite survival. Nippostrongylus brasiliensis was included in the study as an example of unsuccessful parasitism. MATERIALSANDMETHODS Specific pathogen free male C57BL/6J mice were obtained from the Animal Resources Centre, Murdoch University, Western Australia, Australia and were maintained under barrier conditions throughout the experiments described. Nematospiroides dubius and Nippostrongylus brasiliensis were maintained in C57BL mice or SpragueDawley rats, respectively, as described previously (Price and Turner 1983a). In most experiments, mice were given 250 infective N. dubius larvae orally and satisfactory levels of infection were confirmed by egg counts at the end of each experiment. Alternatively 500-600 infective N. brasiliensis larvae were administered subcutaneously and egg counts were carried out on Day 8 or 9. In the majority of experiments, mice were immunized with 100p-g OA (Sigma, USA; five times crystallized) dissolved in PBS and emulsified with FCA (Commonwealth Serum Laboratories, Melbourne, Australia); this OA-FCA was administered subcutaneously into the footpads and base of tail. For the study of delayed type hypersensitivity (DTH) responses mice were then ear challenged with 10 ~1OA (1 mg/ml in PBS) in the right ear and 10 p,l PBS in the left ear. The ear swelling responses reported are the differences in thickness of the test and control ears (X lO-2 mm) measured after 24 hr with a Moore and Wright
micrometer. These values were compared using a double ended Student t test. Results described in Table I relate to the following alternative regimes. (I) 10 pg OA administered subcutaneously (footpads and base of tail) or intraperitoneally in 4 mg Al(OH), (Amphojel, Wyeth Pharmaceuticals, Australia), followed by ear challenge with 10 pg OA. (2) 100 ug OA emulsified in FIA and injected as described for OA-FCA, followed by ear challenge with IO pg OA. (3) Oxazolone (1% in alcohol) painted on the abdominal skin 5 days before ear challenge, which was achieved by painting one ear with 0.5% oxazolone in acetone:olive oil. (4) Unimmunized mice ear challenged with 100 pg crude saline extract of adult N. dub&. (5) OA-FCA immunized mice ear challenged with 10 p,g PPD from Mycobacterium tuberculosis (Crystalline form, Commonwealth Serum Laboratories, Australia) in 10 +I PBS. (PPD is an antigenic component of FCA.) (6) Unimmunized mice ear challenged with 5 pg LPS (Salmonella typhimurium; Difco Laboratories, USA) in 10 pl PBS. (7) OA-FCA immunized mice ear challenged with OA-pulsed peritoneal cells as described by Price and Holt (1986). The enzyme linked immunosorbent assay and statistical analyses have been described in detail previously (Price and Turner 1983b). Individual sera were diluted on microtitration plates coated with 40 pg OAiml alkaline coating buffer and developed with rabbit antimouse IgG or IgM and sheep antirabbit IgG conjugated with alkaline phosphatase. OD,,, values recorded after incubation with a dinitrophenyl phosphate substrate were compared with those generated by a standard serum pool run on each plate, which was assigned a value of 10,000 units of IgG and 1000 units of IgM. The unit values of the test sera were converted to log, and subjected to a nested two-way analysis of variance. Methods used for the measurement of in vitro lymphoproliferation were described in detail in an earlier publication (Price and Turner 1986a). Briefly, lymphoid organs were removed aseptically, chopped with fine scissors, resuspended in cold “mouse buffered balanced salt solution,” and spun at 400g. Spleen preparations were then purified further by passage through cotton wool columns in “low ionic strength medium” and by incubation in 0.18 M NH&I (10 min, 4 C) to remove red blood cells. Lymph node cell preparations from the paraaortic, inguinal, and popliteal nodes which drain the sites of OA-FCA immunization were prepared by an equivalent method but the treatment with NH,CI and low ionic strength medium was
and ear challenged 7 to 28 days later. The data presented in Fig. 1 represents the ac. cumulation of all results obtained over a 12 100 .--------.. o ----.-- .-.----_-.-------___--_. month period. In every case, the ear 80 00 . swelling response to OA in uninfected mice 4 A I was approximately 20 x low2 mm, and the 60. * A . . difference between N. dubius infected and 40. control values was statistically significant if 20. 7 or more days of infection elapsed prior to immunization (t test, P < 0.01-0.05). No 0 5 10 15 20 25 30 35’%6 pattern could be detected in association Time of ammunwhon relatwe to infecton Idaysl with the time elapsing between immunizaFIG. 1. Ratios of DTH responses (24 hr increases in tion and ear challenge. It should be noted ear thickness) derived from groups of approximately dubius, (0, A) or that mice immunized 0 or 6 days after insix infected mice [Nematospiroides Nippostrongylus brasiliensis, (0), and six control fection had established worms in their inmice immunized with ovalbumin in Freund’s complete testines when ear challenged, but failed to adjuvant 0 to 66 days after infection and ear chalshow suppressed responses. N. brasiliensis lenged 7 to 28 days after immunization. Statistically infection induced only marginal suppressignificant differences are identified with a triangle. sion which waned if immunization was delayed to coincide with worm expulsion (t omitted. Viable counts were made by Trypan blue ex- test, P > 0.05 at all times). Thus, DTH r?clusion, and the cells were resuspended at 3 x 106/ml activity was impaired only in mice carrying in RPM11640containing 0.5% autologous serum, 5 x an established infection at the time of im10m5M 2-mercaptoethanol, Hepes buffer, and antibimunization. otics. Triplicate 200 pl aliquots were cultured in Nunc Microculture plates for 72 hr in the presence of To establish whether the depression of IO- 100 p,g OA, 0.1 pg concanavalin A, 25 ug LPS, or DTH responses was paralleled by a de15 pg PPD. They were then pulsed with 0.5 pCi [3H]thymidine (Amersham Corp., Australia) and har- crease in humoral responses, groups of six mice were immunized with OA-FCA 6 or vested after a further I8 hr. To determine the role of suppressor T cells, selected 17 days after infection with N. dubius mice were injected intraperitoneally with cyclophoslarvae, and were bled, together with uninphamide (Endoxan-Asta, West Germany) at 200 mgikg fected controls, 7, 15, 21, and 28 days later body weight, 48 hr before immunization with OA(Fig. 2). There was no alteration in IgG FCA. production in mice immunized 6 or 17 days The responsive potential of primed lymph node cells from infected mice was also studied by adoptive after infection (analysis of variance, P > transfer to recipients given whole body irradiations 0.05), but IgM production was enhanced in (600 rad) from a 300 Kev Seimans Deep X-ray ma- the Day 6 group (analysis of variance, P < chine 24 hr before cell transfer (Table II). Recipients were given OA-FCA (subcutaneously) within an hour 0.005). DTH responses measured in these of the infusion of cells. Cell suspensions were pre- mice were depressed relative to control pared and counted as described for the lymphoprovalues as described in Fig. 1. liferation assay. We also confirmed that N. brasiliensis did not affect humoral responses to OARESULTS FCA and that mice immunized with OAGroups of approximately six mice in- FIA or OA-Al(OH), produced normal titres fected with 200-300 Nematospiroides irrespective of prior infection with N. dubius larvae or 500-600 Nippostrongylus dubius or N. brasiliensis. brasifiensis larvae or retained as uninfected To determine whether the lesion in DTH controls were immunized with OA-FCA responsiveness induced by N. dubius inINFKTEDCONTROL
I%1 120. l
PRICE AND TURNER
6j I Time after
15 n lmmunwztion (days)
FIG. 2. Humoral responses (Log, geometric mean titers to ovalbumin) produced by groups of six mice immunized with ovalbumin in Freund’s complete adjuvant (0) or 17 (A) days after infection with Nemarospiroides dubius. Uninfected immunized controls are denoted (0).
fection lay in the afferent or efferent arm of the response, experiments were performed in which one or the other of these was modified or bypassed. Details of the immunization and challenge schedules are given under Materials and Methods. Results obtained with mice immunized 14 to 21 days after oral infection with N. dubius larvae are summarized in Table I. The first experiment demonstrated that infected mice produced clear DTH responses to N. dubius extract, which were not seen in uninfected mice. Responses of unprimed mice to ear challenge with LPS and contact sensitivity responses to oxazolone were not affected by N. dubius (Experiments 2 and 3). Experiments 4 to 8 demonstrated that immunosuppression was evident whenever DTH responses were induced in the presence of an adjuvant (FCA, FIA, or AI(OH the significance of the
depression being roughly proportional to the magnitude of the control response. Responses elicited by PPD in OA-FCA primed mice were affected in the same way as responses to OA. DTH responses induced by OA-FIA were not affected by N. dubius or N. brasiliensis larvae administered only 7 days before priming. In Experiment 9, control and longterm N. dubius infected mice were immunized with OA-FCA (subcutaneously) and challenged with 5 x lo5 OA pulsed peritoneal cells. This, protocol should circumvent depression associated with antigen loss or defective lymphokine production at the ear test site. Depression equivalent to that generated by challenge with OA was observed (t test, P < 0.002). From these results, it appears that the depression of DTH responsiveness is associated with the generation of T cells involved in the DTH response or suppressor T cells in the draining lymph nodes on stimulation with an adjuvant, rather than to depressed reactions at the ear test site. Since the data just described suggested that N. dubius does not influence the ability to raise a hypersensitivity response in the ear, in vitro antigen driven and adjuvant driven proliferative responses were studied in the lymph nodes draining the site of immunization and in the spleen. The mean cell yields from the draining lymph nodes declined to 30% of control values in mice immunized during the second week of infection, which coincided with the depression of DTH reactivity. However, in the third week, the cell yields normalized while the DTH responses remained low (unpublished results). The degree of antigen priming achieved in the nodes and spleen was then assessed by the in vitro proliferative response to OA (Fig. 3). In Experiment 1, popliteal and paraaortic nodes were collected separately from normal and infected mice (N. dubius, Day - 17), 7 days after immunization with OA-FCA. Whilst cells from the popliteal
TABLE I Responsiveness in the Delayed Type of Hypersensitivity (DTH) of Mice Infected with Nematospiroides
DTH responses” Agents administered Immunization
1 None None Oxazolone sensitization OA-FCA (sc)~ 5 OA-FCA (SC) 6 OA-FIA (sc)~ 7 OA-AI( (SC) 8 OA-AI( (ip)d 9 OA-FCA (SC)
100 pg N. dubirts extract 5 pg LPSf 0.5% Oxazolone 10 pg OA 10 pg PPD’ IO pg OA IO pg OA IO pg OA 5 x IO5OA-pulsed peritoneal cells
2 3 4
252 16 t 3 IS 4 4 29 4 2 13 2 2 I5 2 3 92-3 421
25 2 16 !I 14 2 16 2 622 10 + 7k3 2-1-I
23 ? 2
17 k I
3 2 I 2 2
Note. DTH responses calculated from groups of 6 to I2 mice, where infected mice received 250 N. dubius larvae orally 14 to 21 days before immunization. 0 Arithmetic mean 24-hr ear swelling (k standard error). b Ovalbumin (OA) administered subcutaneously with Freunds complete adjuvant (FCA). c OA administered subcutaneously with Freunds incomplete adjuvant (FIA). d OA administered intraperitoneally with aluminium hydroxide. e Purified protein derivative (PPD) of Mycobrrcterium t/rbercu/osis. J Lipopolysaccharide.
nodes underwent more rapid proliferation on stimulation with OA, the parasite caused minor suppression with higher doses of antigen in cultures from both nodes (100 p.g: t test, P < 0.05). To establish whether the persistence of the OA induced proliferative response or the dispersion of activated cells to the spleen were affected by N. dubius, cell preparations from a pool of the draining nodes and from the spleen were compared later in the response (Experiment 2). The responses of lymph node cells collected 14 days after immunization increased proportionately with the duration of N. dubius infection (in parallel with the decrease in DTH responses), while the responses of splenocytes remained low in all groups. By 25 days after immunization (Experiment 3), the proliferative responses of spleen cells were equivalent to those of the nodes, but the parasite still caused significant increases in responsiveness. It is a useful comparison to note that N. dubius reduced
the yield of cells from the pooled nodes in Experiment 3 but not in Experiment 2, as the specific OA proliferative responses followed a common pattern in these two experiments. During the course of an infection, there was a parallel decrease in the size of nodes collected from mice given OA-Al(OH), (subcutaneously) and OA-driven proliferation was enhanced, as with OA-FCA (Experiment 3). However, as was mentioned earlier (Table I), all DTH responses were low and not significantly reduced by N. dubius in this system. The next aim was to establish the nature of cells in the proliferative response. Although it has been demonstrated that T cells may be the dominant species proliferating in response to local stimulation with an antigen-FCA emulsion (e.g., Corradin et al. 1977), this cannot be assumed for all systems including parasite infected animals. Hence, groups of six control and infected (N. dubius, Day - 18) mice were im-
PRICE AND TURNER 0
Expl -day7 assay ‘-0.
,~ EXQ3-day ZS assay
. Poded nodes
60 %FIG. 3. Mean ovalbumin (OA) specific, lymphoproliferative responses (DPM x 10-3) derived from triplicate 200 ELIcultures of spleen or Iymph node cells from groups of five mice immunized (subcutaneously with OA in Freund’s complete adjuvant or Al(OH), on Day 0 (i.e., 7, 14, or 25 days prior to assay). Key: Nematospiroides dubius, Day - 17 (0); N. dubius Day -7 (A); N. dubius, Day 0 (A); no infection (0). Responses marked with a star (*) are significantly different (t test, P < 0.05-0.001) from the appropriate controls.
munized with OA-FCA (subcutaneously). Cells suspensions prepared from these animals 17 days later were treated with anti412 serum and guinea pig complement or complement alone and cultured with OA, PPD, or concanavalin A (Table II). Responses induced by all three agents in cells from both groups of mice were anti-+,,sensitive. This finding was confirmed with a range of doses of OA and PPD and was
equivalently true for spleen and lymph node cells. The anti-OA responses were low in this experiment because culture was delayed until Day 17 which is optimal for the measurement of responses to PPD. At this time, they are higher in infected than control mice (c.f. Fig. 3). To determine whether cyclophosphamide affected lymphoproliferative responses induced by OA-FCA, groups of six control
dubius, Nippostrongylus TABLE
T-Cell Involvement in the Antigen Specific Proliferative Response Control mice anti-+,, + C’ No mitogen OA” PPDb Con AC
12 2 k 12 3 i
0.3 1.1 0.4 1.7
Untreated No mitogen OA
PPD Con A LPSd
5 22 96 93
k 2 _’ ”
0.4 5 24 6
106 k 7
C’ 3 7 47 15
t 2 k 2
0.7 0.5 4 5
Cyclophosphamide treated 8 27 48 84 30
2 2 t 2 k
-70 -98 -96
anti-+,, + C’ 1.6 2.4 2.4 7.0
k ” 2 t
0.3 1.0 1.1 1.8
3 2 0.5 16 2 0.7 54 k 11 81 z!z10
-85 -96 -91
+22 -50 -9 -71
8 2 0.2 28 2 3 92 k 11 131 2 15 95 AI 6
7 e 0.9 19 2 1 31 ” 2 78 IL 4 26 k 0.4
-31 -66 -41 -72
0.3 5 7 9 1
Note. Lymphoproliferative responses (arithmetic mean DPM x 10u3 2 standard error) derived from triplicate 200 pl cultures of lymph node cells from mice infected with Nematospiroides dubius (Day - 18) and appropriate controls. In the first experiment mice, were sacrificed 17 days after immunization with OA-FCA and suspensions were preincubated with anti+,, and complement (C’) as indicated. In the second experiment, cyclophosphamide (200 mg/kg) was administered on Day - 2 and mice received OA-FCA on Day 0 and were killed on Day 9. LI40 ug ovalbumin (OA) per culture. b 15 ug purified protein derivative (PPD) per culture. c 0.1 u,g concanavalin A (Con A) per culture. d 25 ug lipopolysaccharide (LPS) per culture.
and N. dubius-infected (Day - 15) mice were treated with cyclophosphamide. They were immunized with OA-FCA (subcutaneously) 2 days later and were sacrificed after a further 8 days. Their spleens and popliteal lymph nodes were cultured with OA, PPD, concanavalin A, or LPS. LPS was included to determine whether the infection influenced previously established suppressive effects of cyclophosphamide with respect to B cell functions. However, in this system, responses to LPS were equivalently suppressed in normal and infected mice. In contrast, cyclophosphamide had a greater effect on cells from infected mice than those from controls following stimulation with OA, PPD, or concanavalin A. Since these three agents stimulated largely T cell proliferative responses in normal and infected mice, this result suggests that cyclophosphamide removed a subpopulation
of T cells (possibly suppressor T cells), leaving sufficient T cells (perhaps those involved in DTH responses) to produce the observed proliferation. This would support the contention that infected mice have a greater proportion of suppessor T cells than controls. The ability of lymph node cells from control and infected mice to reconstitute the delayed type hypersensitivity responsiveness of irradiated hosts was studied to establish whether the depressed DTH reactivity identified in mice harboring N. dubius was due to a failure in the generation of the relevant T cells or to excessive production of the equivalent suppressor T cells. Normal and N. dubius infected or N. brasifiensis infected mice were immunized with OA-FCA. Their DTH responses to OA were measured after 8 or I1 days, where-
PRICE AND TURNER
TABLE III The Transfer of Responsiveness to the Delayed Type of Hypersensitivity (DTH) to Irradiated Recipient Mice
Donors Expt 1 Uninfected N. dubius (Day -66)’ None Expt 2 Uninfected N. dubius (Day - 6) N. brasiliensis (Day - 6) N. dubius (Day - 35) None
Donor cell yield (x 10-q
Recipientb 1gG titer
I.5 5.1 -
30 2 4 16 f 3 -
22 2 5 25 t 3 <2
12.6 f 0.5 13.9 f 0.3 3.0 t 0.4
3.1 4.0 4.3 3.6 -
21 k 3 22 k 3 14 2 4 953 -
20 2 22 k 20 -c 24 ” 2?2
12.4 2 14.0 2 12.0 ? 13.7 2 4.7 k
3 4 3 3
0.2 0.4 0.4 0.1 0.5
Note. DTH and IgG responses induced in irradiated mice given 3 x 10’ lymph node cells from control or infected syngeneic donors immunized with ovalbumin-Freunds complete adjuvant (OA-FCA) 8 (Experiment I) or 11 (Experiment 2) days before sacrifice. Recipients received OA-FCA (subcutaneously) within an hour of reconstitution and were bled and challenged for DTH measurement IO days later. 0 Arithmetic mean ear swelling response (2 standard error). b Log, geometric mean IgG titer (2 standard error). c Day of infection (300 Nematospiroides dubircs larvae, oral, or 500 Nippostrongylus brasiliensis larvae, SC).
DISCUSSION upon they were killed and 3 x 10’ draining lymph node cells (pooled paraaortic, inThe experiments described demonstrate guinal, and popliteal) were given intravenously to groups of syngeneic irradiated re- that infection with Nematospiroides dubius cipients, which were subsequently chal- leads to the depression of DTH responses lenged with OA-FCA. The DTH responses induced by OA-FCA. The phenomenon deproduced by the donors and recipients are veloped at the time when the worms ceased shown in Table III. In the donors, N. to be encysted in the duodenal wall and enbrasid~bius infections initiated on Day - 35 or tered the lumen. Nippostrongylus Day -66 significantly depressed DTH re- liensis induced only marginal suppression, sponsiveness (t test, P < 0.05), but neither again while adult worms were in the intesN. dubius nor N. brasiliensis larvae given tine, but the effect was lost if rejection on Day - 6 had any significant effect. In began before the mice were immunized. contrast, all recipient DTH and IgG re- This suggests that the suppression of DTH sponses were normal or marginally in- responsiveness by N. dubius may be mechcreased by N. dubius, although no changes anistically linked with a system which in the DTH responses were statistically sig- permits N. dubius to remain in the host nificant. In Experiment 2, recipient IgG re- when N. brasiliensis would have been responses were significantly elevated when jected. While DTH responsiveness has not been donors were infected 6 days (t test, P < 0.002) or 35 days (P < 0.01) prior to immu- studied previously in mice infected with N. nization. Donor cell yields were slightly re- dubius, similar phenomena have been idenduced by N. dubius in Experiment 1 but tified in other systems. For example, many not Experiment 2, so this cannot be crucial parasites, e.g., Ascaris suum (Crandall and to the results. Crandall 1976) and Trypanosoma brucei
(Askonas et al. 1979), depress footpadswelling responses to sheep erythrocytes administered intravenously but not subcutaneously. Measurement of contact sensitivity to oxazolone in the presence of these parasites can yield normal responses as it did in our system (Table I). The studies listed above suggested that suppression is achieved when the antigen is given intravenously and hence goes initially to the spleen rather than to the peripheral lymph nodes, as it would following subcutaneous or dermal challenge (Kar 1980). The phenomenon is probably linked to the suppressive effects of splenic macrophages implicated in the effects of protozoan parasites (e.g., Wellhausen and Mansfield 1980). There are a relatively small number of reports dealing with responses to antigen given with FCA, none of which deal directly with N. dubias. Active filarial infections depress the DTH responses of jirds to homologous antigens given in FCA, but not to heterologous antigens (Kwa and Mak 1980). Equivalent results have been reported with DTH and lymphoproliferative studies of human tilariasis (e.g., Grove and Forbes 1979), but this parasite is atypical in inducing predominantly specific immunodepression. For example, specific antiparasite responses are easily measured during infections with N. dubius (Price and Turner 1986b), Trichinella spiralis (Cranda11and Crandall 1972), and TYypanosoma cruzi (Cunningham et al. 1981). Depressed reactivity to tuberculin skin tests has been reported in patients with leishmaniasis (Rees et al. 1981) and may reflect mechanisms related to a failure of an experimental animal to respond to FCA. Mackenzie et al. (1979) demonstrated that rats infected with T. brucei and treated with myelin basic protein in FCA were less inclined to develop experimental allergic encephalitis than controls and showed reduced DTH responses to PPD. In view of this report, DTH responses to PPD were
measured in the present system and were found to be depressed (Table I). The initial aim of investigations into the mechanisms responsible for the depression of DTH responses by N. dubirts was to establish whether the lesion lay in the inductive phase in the lymph nodes draining the site of immunization or in the hypersensitivity response in the ear. Responses induced with LPS or by sensitization not involving a potent adjuvant (oxazolone or N. dubius antigen) were not depressed by the parasite (Table I). Mice immunized less than 7 days after infection harboured adult worms at the time of ear test but not at immunization, and responded as well as controls (Fig. I). However, infected mice ear challenged with antigen pulsed peritoneal cells to circumvent defects in antigen presentation or lymphokine production at the ear test site (Table I) produced depressed responses. Taken together, these findings suggest that the lesion lies in the inductive phase of the response in the draining lymph nodes. The next aim was to distinguish between a failure to generate positive effector cells for the DTH reaction and an increase in the generation of the equivalent suppressor T cells. It appeared initially that the size of adjuvant stimulated lymph nodes was reduced by N. dubius infection. This suggested a broad spectrum depression of the proliferative response to FCA. However, long term infected mice developed optimal cell yields while their DTH remained low. A second possibility is that the priming of T cells within the nodes was impaired. However, humoral (T-helper dependent) and anti-&,-sensitive specific lymphoproliferative responses were normal, so clearly all T cells were not affected. Furthermore, irradiated recipients transfused with a fixed number of cells developed normal to elevated delayed type hypersensitivity responses, suggesting that the development of the relevant T cells was not selectively impaired by N. dubius.
Therefore, the possibility that N. dubius increased the level of suppressor T cells was considered. This is consistent with the maintenance of humoral responses, as they are regulated by distinct subpopulations of suppressor T cells (Whisler and Stobo 1978). If N. dubius increased the base levels of suppressor T cells or provided conditions in which their generation in response to a specific stimulus was facilitated, then the adjuvanticity of FCA could lead to the production of an enlarged pool of suppressor T cells in infected mice. The generation of suppressor activity by BCG has been reported previously. For example, Reinisch et al. (1976) showed that C57BL mice treated with FCA generated high levels of helper T cells for IgG production and suppressor T cells for cytotoxic T cell responses. The generation of increased numbers of OA-specific suppressor T cells by FCA in infected mice would be consistent with the high levels of anti-+,, and CY sensitive, OA specific lymphoproliferation demonstrated in spleen and lymph node preparations from such animals. It is also possible that the parasite might induce suppressor T cells with isotype (i.e., DTH) specificity but no antigen specificity. In this circumstance, one must assume that the OA induced lymphoproliferative response does not reflect the DTH reactive potential of the animals. Wren and coworkers (1983) demonstrated reduced graft-versus-host potential and mitogen reactivity in the spleens of C57BL mice treated with BCG. This effect was sensitive to indomethacin and hence may have been mediated by prostaglandins derived from activated macrophages. However, the depression of DTH reactivity by N. dubius developed after the suppression of antibody responses by peritoneal macrophages had subsided one week after infection (Price and Turner 1984). Furthermore, treatment of normal and long term infected mice with indomethacin (c.f. Wren et al. 1983) did not alleviate the suppression of
DTH responses to OA or PPD (unpublished results). Prostaglandins are implicated in the macrophage mediated effect of short term infections, but this was associated with impaired OA induced lymphoproliferation (Price and Holt 1986) which was not observed in the present system. Superficially, the observed optimal responses of irradiated mice reconstituted from infected donors does not support any role for suppressor T cells. However, if the activity of these cells requires cell-to-cell contact or can otherwise be thwarted by dilution in an adoptive host, one would obtain the observed results. It is also possible that the suppressor cells were not in the spleen or draining nodes or were not equivalently active in a secondary response, such as that evoked in this arrangement. Reinisch et al. (1976) demonstrated that suppressor T cells which inhibited primary cytotoxic responses did not affect the responses of primed cells. In conclusion, we have identified a response which was suppressed by a persistent intestinal infection with Nematospiroides dubius, but not by the larval phases of the same parasite or by an acute infecbrasiliensis. tion with Nippostrongyloides The suppression probably acted at the inductive phase of the response in the draining lymph nodes, rather than at the ear test site. However, antibody production and the generation of cells able to proliferate in vitro or to mount adoptive DTH responses in radiation chimeras were not depressed. We consider it most likely that the observed depression is due to suppressor T cells specific to DTH responses generated by the parasite and perhaps locally important for its survival, which are clonally expanded by an adjuvant such as FCA sufficiently for suppression to be manifested systematically. ACKNOWLEDGMENTS This work was supported by the Children’s Medical Research Foundation. This is Publication No. 247 of the Clinical Immunology Research Unit of the Prin-
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